Oncokinase fusion polypeptides associated with hyperproliferative and related disorders, nucleic acids encoding the same and methods for detecting and identifying the same

ABSTRACT

Oncokinase fusion polypeptides associated with hyperproliferative disorders and the polynucleotides encoding for such fusion polypeptides are provided. The fusion polypeptides have a C-terminal tyrosine kinase domain fused to an N-terminal domain that is not normally fused to the C-terminal tyrosine kinase domain and they possess constitutively activated tyrosine kinase activity. Also provided are methods for detecting and identifying the fusion polypeptides and polynucleotides and methods of diagnosing disease conditions associated with the fusion polypeptides and polynucleotides. In addition, screening assays for identifying agents useful for treating disease conditions associated with such fusion polypeptides and polynucleotides are provided. Furthermore, methods of treating disease conditions associated with the presence of the fusion polypeptides are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priorityto the filing date of the U.S. Provisional Patent Application Serial No.60/402,330 filed Aug. 9, 2002 and U.S. Provisional Patent ApplicationSerial No. 60/440,491 filed Jan. 16, 2003; the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is directed to novel oncokinase fusionpolypeptides associated with hyperproliferative disorders and to thepolynucleotides that encode for such fusion polypeptides. This inventionis also directed to methods of identifying and characterizing suchfusion polypeptides and polynucleotides; to methods of diagnosingdisease conditions associated with such fusion polypeptides andpolynucleotides; and to screening assays for identifying agents usefulfor treating disease conditions associated with such fusion polypeptidesand polynucleotides.

[0004] 2. State of the Art

[0005] An accumulation of genetic changes underlies the development andprogression of hyperproliferative disorders, such as cancer, resultingin cells that differ from normal cells in their behavior, biochemistry,genetics, and microscopic appearance. Mutations in DNA that causechanges in the expression level of key proteins, or in the structuresand biological activities of proteins, are thought to be at the heart ofcancer. For example, cancer can be triggered when genes that play acritical role in the regulation of cell growth and survival undergomutations that lead to their over-expression and/or activation. Such“oncogenes” are involved in the dysregulation of growth that occurs incancers.

[0006] Kinases and phosphatases are enzymes involved in phosphorylationand dephosphorylation that help regulate many cellular activities,particularly signaling from the cell membrane to the nucleus to initiatethe cell's entrance into the cell cycle and to control other functions.For example, phosphorylation is important in signal transductionmediated by receptors via extracellular biological signals such asgrowth factors or hormones. Many oncogenes are kinases or phosphatases,i.e. enzymes that catalyze protein phosphorylation or dephosphorylationreactions. Kinases and phosphatases may themselves be specificallyregulated by phosphorylation. A kinase or phosphatase can have itsactivity regulated by one or more distinct kinase or phosphatases,resulting in specific signaling cascades.

[0007] Despite a long-standing need to understand and discover methodsfor regulating cells involved in various disease states, the complexityof signal transduction pathways has been a barrier to the development ofproducts and processes for such regulation. Accordingly, there is a needin the art for improved methods for detecting and modulating theactivity of genes involved in signal transduction and cell cycleregulation and for treating diseases associated with cancer and relateddisease conditions resulting from abnormal phosphorylation activity,e.g., kinase activity.

SUMMARY OF THE INVENTION

[0008] Oncokinase, particularly tyrosine kinase, fusion polypeptidesassociated with hyperproliferative disorders, as well as nucleic acidsencoding the same, are provided. A feature of the subject fusionpolypeptides is that they include a C-terminal tyrosine kinase domainfused to an N-terminal domain that is not normally fused to theC-terminal tyrosine kinase domain, where the subject fusion polypeptidespossess constitutively activated tyrosine kinase activity; i.e., they donot require the presence of an exogenous factor, e.g., a growth factor,to express their catalytic activity. The subject fusion polypeptides arefurther characterized in that they include at least one of the followingfeatures: (a) the C-terminal domain is from a chromosome 4 tyrosinekinase; (b) the N-terminal domain is from a chromosome 4 encodedprotein, e.g., it is a NM_(—)030917 domain; and (c) the fusion proteindoes not arise from a translocation event, i.e., it does not arise fromthe exchange of DNA between different chromosomes; where in certainembodiments, two or more, including all three of, these features arepresent in the subject fusion polypeptides. Also provided are methods ofidentifying and characterizing the subject fusion polypeptides. Alsoprovided are methods of diagnosing disease conditions by detecting thepresence of the subject polypeptides/polynucleotides and/or detectingthe deletions of one or more genomic sequences, where the deletionsresult from the chromosomal deletion event that gives rise to thesubject polypeptides/polynucleotides. In addition, screening assays foridentifying agents that find use in treating disease conditionsassociated with the presence of the subject fusion polypeptides areprovided. Furthermore, methods of treating disease conditions associatedwith the presence of the subject fusion polypeptides are

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows data for a viability assay using imatinib mesylate(Gleevec™) against three leukemia cell lines.

[0010]FIG. 2 provides a diagram illustrating the genetic rearrangementthat gives rise to the oncogene and fusion polypeptide of the subjectinvention.

[0011]FIGS. 3A and 3B provide sequence data of fusion points found inNM_(—)030917-PDGFRα fusion protein coding sequences found in twodifferent EOS patients.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Oncokinase, particularly tyrosine kinase, fusion polypeptidesassociated with hyperproliferative disorders, as well as nucleic acidsencoding the same and methods for detecting and identifying the same,are provided. A feature of the subject fusion polypeptides is that theyinclude a C-terminal tyrosine kinase domain fused to an N-terminaldomain that is not normally fused to the C-terminal tyrosine kinasedomain, where the subject fusion polypeptides possess constitutivelyactivated tyrosine kinase activity. The subject fusion polypeptides arefurther characterized in that they include at least one of the followingfeatures: (a) the C-terminal domain is from a chromosome 4 tyrosinekinase; (b) the N-terminal domain is from a chromosome 4 encodedprotein, e.g., a NM_(—)030917 domain; and (c) the fusion protein doesnot arise from a translocation event, i.e., it does not arise from theexchange of DNA between different chromosomes; where in certainembodiments, at least two of, including all three of, these features arepresent in the subject fusion polypeptides. Also provided are methods ofidentifying and characterizing the subject fusion proteins. Alsoprovided are methods of diagnosing disease conditions by detecting thepresence of the subject polypeptides/polynucleotides and/or detectingthe deletions of one or more genomic sequences, where the deletionsresult from the chromosomal deletion event that gives rise to thesubject polypeptides/polynucleotides. In addition, screening assays foridentifying agents that find use in treating disease conditionsassociated with the presence of the subject fusion polypeptides areprovided. Furthermore, methods of treating disease conditions associatedwith the presense of the subject fusion polypeptides are provided.

[0013] Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0014] In this specification and the appended claims, the singular forms“a,” “an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs. Although any methods, devices and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods, devices and materialsare now described.

[0015] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0016] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the elements thatare described in the publications which might be used in connection withthe presently described invention.

[0017] In further describing the subject invention, the subjectoncokinase fusion polypeptide and nucleic acid compositions aredescribed first in greater detail, followed by a more detailed review ofthe subject antibody, diagnostic, screening and therapeutic embodimentsof the subject invention.

[0018] Oncokinase Fusion Polypeptide Compositions

[0019] As summarized above, the subject invention provides onco,particularly tyrosine, kinase fusion proteins that exhibit constitutivetyrosine kinase activity, i.e., constitutively active kinase fusionpolypeptides. By constitutively active kinase activity is meant that thekinase activity is “always on,” under intracellular conditions, asdetermined using the assay described in Science et al., (1998)279:577-580.

[0020] A feature of many embodiments of the subject fusion proteins isthat they confer an immortalized, and often hyperproliferative,phenotype onto a cell in which they are present. In other words, cellsthat express the subject fusion proteins are ones that have animmortalized and often hyperproliferative phenotype. By “immortalized”is meant that the cell is immortal as determined using the assaydescribed in Lab. Invest. (2002) 82:323-333. By “hyperproliferative” ismeant that the cell divides at an above normal rate, as determined usingthe assay described in Cancer Cell. (2002) 1: 421-432.

[0021] The subject fusion proteins are characterized by having aC-terminal tyrosine kinase domain which is fused, either directly orthrough a linking domain, to an N-terminal domain that is from adifferent protein, i.e., is not from the same protein as the proteinfrom which the C-terminal tyrosine kinase is obtained. In certainembodiments, the fusion of the N-terminal domain to the C-terminaltyrosine kinase domain leads to or provides for the kinase domain beingconstitutively active, as described above.

[0022] A further characteristic of the subject fusion polypeptides isthat they also include at least one of the following features:

[0023] (1) they have a C-terminal chromosome 4 tyrosine kinase domain(i.e., a chromosome 4 encoded tyrosine kinase encoded by a codingsequence found on chromosome 4);

[0024] (2) they have an N-terminal domain of a chromosome 4 protein(i.e., a chromosome 4 encoded protein encoded by a coding sequence foundon chromosome 4), e.g., NM_(—)030917; and

[0025] (3) they do not arise from a translocation event involvingexchange of genetic information between different chromosomes.

[0026] In certain embodiments, the fusion polypeptides include at leasttwo of the above features, and in certain of these embodiments thefusion peptides include all three of the above features, e.g., both theC- and N-terminal domains are from chromosome 4 encoded proteins.

[0027] By chromosome 4 tyrosine kinase is meant a tyrosine kinase whosegenomic coding sequence is located on the human chromosome 4. Thechromosome 4 tyrosine kinase domain of the subject fusion proteins mayinclude a domain or portion of a number of different chromosome 4tyrosine kinases, where representative chromosome 4 tyrosine kinases ofinterest include: PDGFRα, c-Kit and VEGFR-2. In many embodiments, thechromosome 4 tyrosine kinase is PDGFRα.

[0028] The fusion polypeptides of the subject invention typicallyinclude only a portion of the chromosome 4 tyrosine kinase, such thatthey do not include the entire coding sequence for the chromosome 4tyrosine kinase. The portion is typically a C-terminal portion or domainof the chromosome 4 tyrosine kinase, which portion or domain exhibitskinase activity. The length of the portions or domains present in thesubject fusion polypeptides is typically at least about 30% smaller,usually at least about 40% smaller and more usually at least about 50%smaller (in terms of residue number) than the full-length chromosome 4tyrosine kinase of which it is a portion. In many embodiments, thelength of the C-terminal chromosome 4 tyrosine kinase domains found inthe subject fusion polypeptides is at least about 400 residues, usuallyat least about 450 residues and more usually at least about 500residues, where the length of this C-terminal domain typically ranges inmany embodiments from about 400 to about 1500, usually from about 500 toabout 1200 and more usually from about 500 to about 1000 residues.

[0029] As summarized above, the fusion polypeptides of the presentinvention include an N-terminal domain of a protein that, when presentin the subject fusion proteins, results in the C-terminal kinase domainbeing constitutively active. Accordingly, the N-terminal domain may beconsidered to be a kinase activating protein. The N-terminal domain is,in many embodiments, a domain of a chromosome 4 protein, where thechromosome 4 protein is typically chromosomally located within proximityto the chromosome 4 tyrosine kinase from which the C-terminal of thefusion protein is derived. Since the N-terminal chromosome 4 protein islocated in proximity, the distance separating its genomic codingsequence from that of the genomic coding sequence of the tyrosine kinasetypically does not exceed about 10 million base pairs, usually does notexceed about 5 million base pairs and more usually does not exceed about3 million base pairs.

[0030] In certain embodiments, the N-terminal domain of the subjectfusion polypeptides is a domain or portion of the chromosome 4 proteinwhich is encoded by the gene with the Genbank accession no.NM_(—)030917, i.e., a “NM_(—)030917 protein”. In some priorpublications, the NM_(—)030917 gene was identified by the Genbankaccession no. BC017724. This gene has recently been named Fip1L1 (seeCools et al., N. Eng. J. Med. (2003) 348:1201-1214; and has also beennamed Rhe (See Griffin et al., Proc. Nat'l Acad. Sci. USA (2003) 100:7830-7835.

[0031] The fusion polypeptides of the subject invention typicallyinclude only a portion of the chromosome 4 kinase activating protein,such that they do not include the entire amino acid sequence of theprotein. The portion is typically an N-terminal portion or domain of theprotein, which, when fused to a C-terminal kinase domain in the fusionprotein, leads to constitutive activation of the kinase domain of thefusion protein. The length of the portions or domains present in thesubject fusion polypeptides is typically at least about 1% smaller,usually at least about 5% smaller and more usually at least about 10%smaller (in terms of residue number) than the full-length chromosome 4protein of which it is a portion. In many embodiments, the length of theN-terminal chromosome 4 kinase activating domains found in the subjectfusion polypeptides is at least about 50 residues, usually at leastabout 100 residues and more usually at least about 200 residues, wherethe length of this N-terminal domain typically ranges in manyembodiments from about 50 to about 350, usually from about 200 to about350 and more usually from about 300 to about 350 residues.

[0032] The subject fusion proteins range in length from about 500 toabout 2000 residues, usually from about 700 to about 1500 residues andmore usually from about 800 to about 1200 amino acid residues, and theprojected molecular weight of the subject proteins based solely on thenumber of amino acid residues in the protein ranges from about 55 toabout 220, usually from about 77 to about 165 and more usually fromabout 88 to about 132 kDa. As the subject fusion proteins may bemodified, e.g., phosphorylated, or modified in alternative ways, theactual molecular weight of these proteins may be substantially higherthan the above projected molecular weights, typically ranging from about1.1 to about 2.0 times higher than the projected molecular weight.

[0033] Of particular interest in certain embodiment are fusion proteinsthat have an amino acid sequence that is substantially the same as, oridentical to, the sequence appearing as SEQ ID NOs: 01, 02, 03 or 04, asprovided below.

[0034] By “substantially the same as” is meant a protein having asequence that has at least about 50%, usually at least about 60% andmore usually at least about 75%, and in many embodiments at least about80%, usually at least about 90% and more usually at least about 95%,96%, 97%, 98% or 99% sequence identity with the sequence of the aboveprovided sequences, as measured by the BLAST compare two sequencesprogram available on the NCBI website using default settings, asmeasured over the entire length of the protein, where the website hasthe address made up by placing “www.” in front of and “.gov” in back of“ncbi.nlm.nih”.

[0035] In addition to the specific fusion proteins described above,homologs or proteins (or fragments thereof from other species, i.e.,other animal species, are also provided, where such homologs or proteinsmay be from a variety of different types of species, usually mammals,e.g., rodents, such as mice, rats; domestic animals, e.g. horse, cow,dog, cat; and primates, e.g., monkeys, baboons, humans etc. By homologis meant a protein having at least about 35%, usually at least about 40%and more usually at least about 60% amino acid sequence identity to thespecific human fusion protein as identified above, where sequenceidentity is determined using the algorithm described supra.

[0036] In certain embodiments, the fusion proteins of the subjectinvention are present in a non-naturally occurring environment, e.g.,are separated from their naturally occurring environment. In certainembodiments, the subject proteins are present in a composition that isenriched for the subject proteins as compared to the subject proteins intheir naturally occurring environment. As such, purified fusion proteinsaccording to the subject invention are provided, where by purified ismeant that the proteins are present in a composition that issubstantially free of non-fusion proteins of the subject invention,where by substantially free is meant that less than 90%, usually lessthan 60% and more usually less than 50% of the composition is made up ofnon-fusion proteins of the subject invention.

[0037] In certain embodiments of interest, the fusion proteins arepresent in a composition that is substantially free of the constituentsthat are present in its naturally occurring environment. For example, ahuman fusion protein comprising a composition according to the subjectinvention in this embodiment will be substantially, if not completely,free of those other biological constituents, such as proteins,carbohydrates, lipids, etc., with which it is present in its naturalenvironment. As such, protein compositions of these embodiments willnecessarily differ from those that are prepared by purifying the proteinfrom a naturally occurring source, where at least trace amounts of theconstituents or other components of the protein's naturally occurringsource will still be present in the composition prepared from thenaturally occurring source.

[0038] The fusion proteins of the subject invention may also be presentas isolates, by which is meant that the proteins are substantially freeof both non-fusion proteins and other naturally occurring biologicmolecules, such as oligosaccharides, polynucleotides and fragmentsthereof, and the like, where substantially free in this instance meansthat less than 70%, usually less than 60% and more usually less than 50%(by dry weight) of the composition containing the isolated fusionproteins is a non-fusion protein naturally occurring biologicalmolecule. In certain embodiments, the fusion proteins are present insubstantially pure form, where by substantially pure form is meant atleast 95%, usually at least 97% and more usually at least 99% pure.

[0039] In addition to the naturally occurring proteins, polypeptidesthat vary from the naturally occurring proteins are also provided. Bypolypeptide is meant proteins having an amino acid sequence encoded byan open reading frame (ORF) of a fusion protein coding sequence,described below, including the full length protein and fragmentsthereof, particularly biologically active fragments and/or fragmentscorresponding to functional domains, and including fusions of thesubject fusion proteins to yet additional proteins or parts thereof,e.g., immunoglobulin domains, peptididic tags; and the like. Fragmentsof interest will typically be at least about 10 aa in length, usually atleast about 50 aa in length, and may be as long as 300 aa in length orlonger, but will usually not exceed about 1000 aa in length.

[0040] Nucleic Acid Compositions

[0041] Also provided are nucleic acid compositions that encode thesubject fusion polypeptides and fragments thereof, etc., as describedabove. Specifically, nucleic acid compositions encoding the subjectpolypeptides, as well as fragments or homologs thereof, are provided. By“nucleic acid composition” is meant a composition comprising a sequenceof nucleotide bases that encodes a fusion polypeptide according to thesubject invention, i.e., a region of genomic DNA capable of beingtranscribed into mRNA that encodes a fusion polypeptide of the subjectinvention, the mRNA that encodes and directs the synthesis of a fusionpolypeptide of the subject invention, the cDNA derived from reversetranscription of the mRNA, etc. Specific nucleic acids of interestinclude those identified herein as SEQ ID NO:05; SEQ ID NO:06; SEQ IDNO:07 and SEQ ID NO:08. Also encompassed in this term are nucleic acidsthat are homologous, substantially similar or identical to the nucleicacids specifically disclosed herein, e.g., SEQ ID NO:05; SEQ ID NO:06;SEQ ID NO:07 and SEQ ID NO:08, where sequence similarity is determinedusing the BLAST compare functionality provided online by the NationalCenter for Biotechnology (using default settings).

[0042] Also provided are nucleic acids that are homologous to theprovided nucleic acids, at least with respect to the coding regionsthereof. The source of homologous nucleic acids to those specificallylisted above may be any mammalian species, e.g., primate species,particularly human; rodents, such as rats and mice, canines, felines,bovines, equines, etc; as well as non-mammalian species, e.g., yeast,nematodes, etc. Between mammalian species, e.g., human and mouse,homologs typically have substantial sequence similarity, e.g., at least75% sequence identity, usually at least 90%, more usually at least 95%between nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403-10 (using default settings, i.e. parameters w=4 and T=17).Unless indicated otherwise, the sequence similarity values reportedherein are those determined using the above referenced BLAST programusing default settings. Of particular interest in certain embodimentsare nucleic acids including a sequence substantially similar to thespecific nucleic acids identified above, where by substantially similaris meant having sequence identity to this sequence of at least about90%, usually at least about 95% and more usually at least about 99%.

[0043] Also provided are nucleic acids that hybridize to theabove-described nucleic acids under stringent conditions. An example ofstringent hybridization conditions is overnight incubation at 42° C. ina solution: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. Stringent hybridizationconditions are hybridization conditions that are at least as stringentas the above representative conditions. Other stringent hybridizationconditions are known in the art and may also be employed to identifynucleic acids of this particular embodiment of the invention.

[0044] Nucleic acids encoding the proteins and polypeptides of thesubject invention may be cDNAs or genomic DNAs, as well as fragmentsthereof. The nucleic acids may also be mRNAs, e.g., transcribed fromgenomic DNA, that encode (i.e. are translated into) the subject proteinsand polypeptides. Also provided are genes encoding the subject proteins,where the term “gene” means the open reading frame encoding specificproteins and polypeptides, and introns that are present in the openreading frame, as well as adjacent 5′ and 3′ non-coding nucleotidesequences involved, e.g., untranslated regions, promoter or otherregulatory elements, etc., in the regulation of expression, up to about20 kb beyond the coding region, but possibly further in eitherdirection. The gene may be introduced into an appropriate vector forextrachromosomal maintenance or for integration into a host genome.

[0045] The term “cDNA” as used herein is intended to include all nucleicacids that share the arrangement of sequence elements found in nativemature mRNA species or the complementary sequences thereof, wheresequence elements at least include exons. Normally mRNA species havecontiguous exons, with the intervening introns, when present, beingremoved by nuclear RNA splicing, to create a continuous open readingframe encoding an oncokinase fusion protein according to the subjectinvention.

[0046] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include specific transcriptionaland translational regulatory sequences, such as promoters, enhancers,etc., including about 1 kb, but possibly more, of flanking genomic DNAat either the 5′ or 3 end of the transcribed region. The genomic DNA maybe isolated as a fragment of 100 kbp or smaller; and substantially freeof flanking chromosomal sequence. The genomic DNA flanking the codingregion, either 3′ or 5′, or internal regulatory sequences as sometimesfound in introns, contains sequences required for proper tissue andstage specific expression.

[0047] The nucleic acid compositions of the subject invention may encodeall or a part of the subject proteins and polypeptides, described ingreater detail above. Double or single stranded fragments may beobtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 nt, usually at least 18 nt or 25 nt,and may be at least about 50 nt.

[0048] The nucleic acids of the subject invention are isolated andobtained in substantial purity, generally as other than an intactchromosome. Usually, the DNA will be obtained substantially free ofother nucleic acid sequences that do not include a fusion proteinsequence or fragment thereof, generally being at least about 50%,usually at least about 90% pure and are typically “recombinant,” i.e.flanked by one or more nucleotides with which it is not normallyassociated on a naturally occurring chromosome.

[0049] In addition to the plurality of uses described in greater detailin following sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the subject polypeptides, asdescribed above.

[0050] Also provided are nucleic acid probes, as well as constructs,e.g., vectors, expression systems, etc., as described more fully below,that include a nucleic acid sequence as described above. Probes of thesubject invention are generally fragments of the provided nucleic acid.The probes may be a large or small fragment, generally ranging in lengthfrom about 10 to 100 nt, usually from about 15 to 50 nt. In using thesubject probes, nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate)(or analogousconditions) and remain bound when subjected to washing at higherstringency conditions, e.g., 55° C. in 1×SSC (0.15 M sodiumchloride/0.015 M sodium citrate) (or analogous conditions). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate)(or analogous conditions). Nucleic acids having aregion of substantial identity to the provided nucleic acid sequencesbind to the provided sequences under stringent hybridization conditions.By using probes, particularly labeled probes of DNA sequences, one canisolate homologous or related sequences.

[0051] The subject nucleic acids may be produced using any convenientprotocol, including synthetic protocols, e.g., such as those where thenucleic acid is synthesized by a sequential monomeric approach (e.g.,via phosphoramidite chemistry); where subparts of the nucleic acid areso synthesized and then assembled or concatamerized into the finalnucleic acid, and the like. Where the nucleic acid of interest has asequence that occurs in nature, the nucleic acid may be retrieved,isolated, amplified etc., from a natural source using conventionalmolecular biology protocols.

[0052] Also provided are constructs comprising the subject nucleic acidcompositions, e.g., those that include a fusion protein coding sequence,inserted into a vector, where such constructs may be used for a numberof different applications, including propagation, screening, genomealteration, and the like, as described in greater detail below.Constructs made up of viral and non-viral vector sequences may beprepared and used, including plasmids, as desired. The choice of vectorwill depend on the particular application in which the nucleic acid isto be employed. Certain vectors are useful for amplifying and makinglarge amounts of the desired DNA sequence. Other vectors are suitablefor expression in cells in culture, e.g., for use in screening assays.Still other vectors are suitable for transfer and expression in cells ina whole animal, e.g., in the production of animal models ofhyperproliferative diseases. The choice of appropriate vector is wellwithin the ability of those of ordinary skill in the art. Many suchvectors are available commercially. To prepare the constructs, thepartial or full-length nucleic acid is inserted into a vector typicallyby means of DNA ligase attachment to a cleaved restriction enzyme sitein the vector. Alternatively, the desired nucleotide sequence can beinserted by homologous recombination in vivo. Typically, homologousrecombination is accomplished by attaching regions of homology to thevector on the flanks of the desired nucleotide sequence. Regions ofhomology are added by ligation of oligonucleotides, or by polymerasechain reaction using primers that include both the region of homologyand a portion of the desired nucleotide sequence, for example. Yetanother means to insert the nucleic acids into appropriate vectors is toemploy one of the increasingly employed recombinase based methods fortransferring nucleic acids among vectors, e.g., the Creator™ system fromClontech; the Gateway™ system from Invitrogen, etc.

[0053] Also provided are expression cassettes that include a codingsequence. By expression cassette is meant a nucleic acid that includes asequence encoding a subject peptide or protein operably linked to apromoter sequence, where by operably linked is meant that expression ofthe coding sequence is under the control of the promoter sequence.

[0054] Preparation of Polypeptides According to the Subject Invention

[0055] The subject fusion proteins may be obtained using any convenientprotocol. As such, they may be obtained from naturally occurring sourcesor recombinantly produced. Naturally occurring sources of the subjectproteins include tissues and portions/fractions, including cells, celllines and fractions thereof, e.g., extracts, homogenates etc., thatinclude cells in which the desired protein is expressed.

[0056] The subject proteins may also be obtained from syntheticprotocols, e.g., by expressing a recombinant gene encoding the subjectprotein, such as the polynucleotide compositions described above, in asuitable host under conditions sufficient for post-translationalmodification to occur in a manner that provides the expressed fusionprotein with the desired constitutively active kinase activity. Forexpression, an expression cassette may be employed. The expressioncassette or vector will provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and under the translationalcontrol of the translational initiation region, and a transcriptionaland translational termination region. These control regions may benative to a gene of the subject invention, or may be derived fromexogenous sources.

[0057] Expression cassettes may be prepared comprising a transcriptioninitiation region, the nucleic acid coding sequence or fragment thereof,and a transcriptional termination region. Of particular interest is theuse of sequences that allow for the expression of functional epitopes ordomains, usually at least about 8 amino acids in length, more usually atleast about 15 amino acids in length, to about 25 amino acids, and up tothe complete open reading frame of the coding sequence. Afterintroduction of the DNA, the cells containing the construct may beselected by means of a selectable marker, the cells expanded and thenused for expression.

[0058] The subject proteins and polypeptides may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. For large scale production ofthe protein, a unicellular organism, such as E. col, B. subtilis, S.cerevisiae, insect cells in combination with baculovirus vectors, orcells of a higher organism such as vertebrates, particularly mammals,e.g. COS 7 cells, may be used as the expression host cells. In somesituations, it is desirable to express the gene in eukaryotic cells,where the encoded protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the completesequence may be used to identify and investigate parts of the proteinimportant for function.

[0059] Specific expression systems of interest include bacterial, yeast,insect cell and mammalian cell derived expression systems.Representative systems from each of these categories are provided below:

[0060] (i) Bacteria

[0061] Expression systems in bacteria include those described in Changet al., Nature (1978) 275:615; Goeddel et al., Nature (1979) 281:544;Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776; U.S.Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA) (1983)80:21-25; and Siebenlist et al., Cell (1980) 20:269.

[0062] (ii) Yeast

[0063] Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

[0064] (iii) Insect Cells

[0065] Expression of heterologous genes in insects is accomplished asdescribed in U.S. Pat. No. 4,745,051; Friesen et al., “The Regulation ofBaculovirus Gene Expression”, in: The Molecular Biology Of Baculoviruses(1986) (W. Doerfler, ed.); EP 0 127,839; EP 0 155,476; and Vlak et al.,J. Gen. Virol. (1988) 69:765-776; Miller et al., Ann. Rev. Microbiol.(1988) 42:177; Carbonell et al., Gene (1988) 73:409; Maeda et al.,Nature (1985) 315:592-594; Lebacq-Verheyden et al., Mol. Cell. Biol.(1988) 8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985)82:8844; Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA(1988) 7:99. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts are described in Luckow et al.,Bio/Technology (1988) 6:47-55, Miller et al., Generic Engineering (1986)8:277-279, and Maeda et al., Nature (1985) 315:592-594.

[0066] (iv) Mammalian Cells

[0067] Mammalian expression is accomplished as described in Dijkema etal., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad. Sci. (USA)(1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S. Pat. No.4,399,216. Other features of mammalian expression are facilitated asdescribed in Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato,Anal. Biochem. (1980) 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866,4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.

[0068] When any of the above host cells, or other appropriate host cellsor organisms, are used to replicate and/or express the polynucleotidesor nucleic acids of the invention, the resulting replicated nucleicacid, RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism.

[0069] Once the source of the protein is identified and/or prepared,e.g., a transfected host expressing the protein is prepared, the proteinis then purified to produce the desired fusion protein comprisingcomposition. Any convenient protein purification procedures may beemployed, where suitable protein purification methodologies aredescribed in Guide to Protein Purification, (Deuthser ed.) (AcademicPress, 1990). For example, a lysate may be prepared from the originalsource, e.g. naturally occurring cells or tissues that express thesubject fusion proteins or the expression host expressing the subjectfusion proteins, and purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and the like.

[0070] Methods of Identifying/Characterizing Onco-Tyrosine Kinase FusionProteins

[0071] Also provided by the subject invention are methods foridentifying and characterizing onco-tyrosine kinase fusion proteins in asample, e.g., a cell, tissue or other sample of interest. In suchmethods, onco-tyrosine kinase fusion proteins are identified by firstscreening the sample of interest to determine whether or not anyonco-tyrosine kinase fusion proteins are present in the sample. Toscreen a sample, tyrosine-phosphorylated proteins are typically firstseparated from the remaining constituents of said sample to produce apopulation of sample derived tyrosine phosphorylated proteins.Separation or isolation of the tyrosine-kinase fusion proteins from theremaining components in the same can be accomplished using anyconvenient protocol, e.g., via immunoprecipitation with ananti-phosphotyrosine antibody. Next, the constituent members of theobtained population of sample derived tyrosine phosphorylated proteinsare evaluated for the presence of domains from two or more differentproteins. In other words, one or more of the different proteins in theisolated population of tyrosine phosphorylated proteins are evaluated todetermine whether they include domains from two or more differentproteins. This evaluating step may be accomplished using any convenientprotocol. In one representative embodiment, the population of tyrosinephosphorylated proteins is separated or fractionated into itsconstituent proteins using, for example, SDS-PAGE, 2-dimensionalIE/PAGE, high-performance liquid chromatography, capillaryelectrophoresis, etc. Next, the constituent proteins are cleaved intosmaller sized peptides, e.g., via subjection to proteolysis using anendoproteinase such as trypsin. The resultant peptides are thenseparated or fractionated using, for example, microbore capillaryelectrophoresis, high-performance liquid chromatography, or massspectrometry. The resultant isolated or fractionated peptides are thensequenced using, for example, automated Edman degradation or massspectrometry. The resultant sequences are then compared to the sequencesof the observed peptides with known or predicted peptide sequences fromproteins expressed in the organism from which the sample was obtained.Next, a determination is made as to which of the constituent proteinsprovides proteolysis peptides from two different proteins, for example,peptides from the N-terminal domain of the protein encoded by geneNM_(—)030917 and peptides from the C-terminal domain of PDGFRA. In thismanner, a sample of interest is screened for onco-tyrosine kinase fusionproteins.

[0072] Any identified onco-tyrosine kinase fusion proteins can befurther characterized to identify one or more of: (a) the full aminoacid sequence; (b) the sequence of an encoding nucleic acid, e.g., mRNAencoding the fusion protein; (c) the sequence of the gene or genomic DNAencoding the fusion protein, etc. For example, the observed peptidesequences of the identified fusion protein can be employed to design PCRprimers that allow for amplification of all or part of an mRNA encodingthe fusion protein, for example, the region of the mRNA that comprisesthe fusion junction. The resultant amplified cDNA encoding all or partof the fusion protein can then be directly sequenced or first cloned andthe sequence of the cloned cDNA encoding all or part of the fusionprotein determined using, for example, automated dideoxy DNA sequencing.The resultant sequence can then be used to determine the cDNA sequenceto predict the sequence of a novel proteolytic peptide encompassing thefusion junction within the fusion protein. The presence of the novelproteolytic peptide encompassing the fusion junction can then bedetermined by for example, mass spectrometry or automated Edmandegradation. The determined cDNA sequence can also be employed to designprimers for amplification of genomic DNA encompassing the fusionjunction. The resultant amplified genomic DNA sequence encompassing thefusion junction can then be directly sequenced or first cloned and thesequence of the cloned genomic DNA determined using, for example,automated dideoxy DNA sequencing. In this way, the identified fusionproteins are further characterized.

[0073] Antibodies

[0074] Also provided are antibodies that bind to the subject fusionproteins and homologs thereof. Suitable antibodies are obtained byimmunizing a host animal with peptides comprising all or a portion ofthe fusion protein. Suitable host animals include rat, sheep, goat,hamster, rabbit, etc. The origin of the protein immunogen may be mouse,rat, monkey etc, but is human in many embodiments. The host animal willgenerally be a different species than the immunogen, e.g. human proteinused to immunize rabbit, etc.

[0075] The immunogen may include the complete protein, or fragments andderivatives thereof; e.g., a fragment comprising the unique sequencefound at the site of fusion of the N- and C-terminal domains. Immunogensemployed in certain embodiments include all or a part of the subjectfusion protein, where these residues contain any post-translationmodifications, such as glycosylation, found on the native targetprotein. Immunogens comprising the fusion protein are produced in avariety of ways known in the art, e.g. expression of cloned genes usingconventional recombinant methods, isolation from HEC, etc.

[0076] For preparation of polyclonal antibodies, the first step isimmunization of the host animal with the target protein, where thetarget protein will preferably be in substantially pure form, comprisingless than about 1% contaminant. The immunogen may include the completetarget protein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil and water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, mice, rats, sheep, goats, andthe like. The target protein is administered to the host, usuallyintradermally, with an initial dosage followed by one or more, usuallyat least two, additional booster dosages. Following immunization, theblood from the host will be collected, followed by separation of theserum from the blood cells. The Ig present in the resultant antiserummay be further fractionated using known methods, such as ammonium saltfractionation, DEAE chromatography, and the like.

[0077] Monoclonal antibodies of the subject invention may be produced byconventional techniques. Generally, the spleen and/or lymph nodes of animmunized host animal provide a source of plasma cells. The plasma cellsare immortalized by fusion with myeloma cells to produce hybridomacells. Culture supernatant from individual hybridomas is screened usingstandard techniques to identify those producing antibodies with thedesired specificity. Suitable animals for production of monoclonalantibodies to the human protein include mouse, rat, hamster, etc. Toraise antibodies against the human protein, the animal will generally bea hamster, guinea pig, rabbit, etc. The antibody may be purified fromthe hybridoma cell supernatants or ascites fluid by conventionaltechniques, e.g. affinity chromatography using MPTS bound to aninsoluble support, protein A sepharose, etc.

[0078] The antibody may be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al. (1994) J. Biol. Chem. 269:26267-73, and others. DNAsequences encoding the variable region of the heavy chain and thevariable region of the light chain are ligated to a spacer encoding atleast about 4 amino acids of small neutral amino acids, includingglycine and/or serine. The protein encoded by this fusion allowsassembly of a functional variable region that retains the specificityand affinity of the original antibody.

[0079] Diagnostic Applications

[0080] Also provided are methods of diagnosing disease states associatedwith fusion protein activity (or even the absence thereof)(such as thosedisease conditions listed below), e.g., based on detecting/observinglevels of fusion protein or the presence and/or expression level of thegene/coding sequence in a biological sample of interest, and/ordetecting the deletions of one or more nucleic acid (particularlygenomic) sequences in a sample of interest, where the deletions resultfrom the chromosomal deletion event that gives rise to the subjectpolypeptides/polynucleotides.

[0081] Samples, as used herein, include biological fluids such as blood,cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like;organ or tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. Samples may also include cells, which may besolitary or in need of being dissociated in the case of solid tissues.Alternatively tissue sections may be analyzed or a lysate of the cellsmay be prepared.

[0082] A number of methods are available for determining the presenceand/or expression level of a gene or protein in a particular sample. Forexample, diagnosis may be performed by a number of methods to determinethe absence or presence or altered amounts of fusion protein in apatient sample. For example, detection may utilize staining of cells orhistological sections with labeled antibodies, performed in accordancewith conventional methods. Cells are permeabilized to stainintracellular molecules. The antibodies of interest are added to thecell sample, and incubated for a period of time sufficient to allowbinding to the epitope, usually at least about 10 minutes. The antibodymay be labeled with radioisotopes, enzymes, fluorophores,chemiluminescers, or other labels for direct detection. Alternatively, asecond stage antibody or reagent is used to amplify the signal. Suchreagents are well known in the art. For example, the primary antibodymay be conjugated to biotin, with horseradish peroxidase-conjugatedavidin added as a second stage reagent. Final detection uses a substratethat undergoes a color change in the presence of the peroxidase.Alternatively, the secondary antibody conjugated to a flourescentcompound, e.g. fluorescein, rhodamine, Texas red, etc. The absence orpresence of antibody binding may be determined by various methods,including flow cytometry of dissociated cells, microscopy, radiography,scintillation counting, etc.

[0083] Alternatively, one may focus on the presence of a gene encodingthe fusion protein and/or expression of the fusion protein. A number ofmethods are available for analyzing nucleic acids for the presence of aspecific sequence, e.g., a coding sequence for the subject fusionproteins. Where large amounts of DNA are available, genomic DNA is useddirectly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis. Cellsthat express the fusion protein may be used as a source of mRNA, whichmay be assayed directly or reverse transcribed into cDNA for analysis.The nucleic acid may be amplified by conventional techniques, such asthe polymerase chain reaction (PCR), to provide sufficient amounts foranalysis. The use of the polymerase chain reaction is described inSaiki, et al. (1985),. Science 239:487, and a review of techniques maybe found in Sambrook, et al. Molecular Cloninq: A Laboratory Manual, CSHPress 1989, pp.14.2-14.33. Alternatively, various methods are known inthe art that utilize oligonucleotide ligation as a means of detectingpolymorphisms, for examples see Riley et al. (1990), Nucl. Acids Res.18:2887-2890; and Delahunty et al. (1996), Am. J. Hum. Genet.58:1239-1246.

[0084] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two-stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

[0085] The sample nucleic acid, e.g., amplified or cloned fragment, maybe analyzed for variations from the specifically provided “wild type”sequences provided herein by one of a number of methods known in theart. The nucleic acid may be sequenced by dideoxy or other methods, andthe sequence of bases compared to a wild-type sequence. Hybridizationwith the variant sequence may also be used to determine its presence, bySouthern blots, dot blots, etc. The hybridization pattern of a controland variant sequence to an array of oligonucleotide probes immobilizedon a solid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

[0086] Screening for variants or mutations may be based on thefunctional or antigenic characteristics of the protein. Proteintruncation assays are useful in detecting deletions that may affect thebiological activity of the protein. Various immunoassays designed todetect polymorphisms in proteins may be used in screening. Where manydiverse genetic mutations lead to a particular disease phenotype,functional protein assays have proven to be effective screening tools.The activity, e.g. kinase functionality, of the encoded protein may bedetermined by comparison with the wild-type protein.

[0087] Diagnostic methods of the subject invention in which the level ofexpression is of interest will typically involve comparison of thenucleic acid abundance of a sample of interest with that of a controlvalue to determine any relative differences, where the difference may bemeasured qualitatively and/or quantitatively, which differences are thenrelated to the presence or absence of an abnormal expression pattern. Avariety of different methods for determining the nucleic acid abundancein a sample are known to those of skill in the art, where particularmethods of interest include those described in: Pietu et al., GenomeRes. (June 1996) 6: 492-503; Zhao et al., Gene (Apr. 24, 1995) 156:207-213; Soares, Curr. Opin. Biotechnol. (October 1997) 8: 542-546;Raval, J. Pharmacol Toxicol Methods (November 1994) 32: 125-127;Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz &Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Hong et al.,Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143:298. Also of interest are the methods disclosed in WO 97/27317, thedisclosure of which is herein incorporated by reference.

[0088] In certain embodiments, the diagnostic applications may furtherinclude a further fusion protein characterization step, where thepresence of the fusion protein of interest is found. For example, onemay further characterize the particular fusion point of the fusionprotein, where knowledge of the particular fusion point is of use, e.g.,in developing rational treatment protocols, as described below. One mayalso further characterize the fusion protein to determine whether it isresistant to any particular contemplated therapeutic agent. For example,the fusion protein may include a mutation or variation as compared tothe “wild-type” sequence that confers resistance to a particularpharmacological agent. A specific representative embodiment of such amutation or alteration is the T674I mutation in the NM_(—)030917-PDGFRαfusion protein, as described in greater detail in the experimentalsection, below.

[0089] In certain embodiments, instead of (or in addition to) detectingthe presence of the subject fusion proteins and/or nucelci acidsencoding the same, as described above, the absence of one or morenucleic acids, e.g., genomic sequences, is detected, where the absenceof the one or more nucleic acids occurs because of a chromosomaldeletion event that results in the presence of the subject fusionproteins, and therefore can be employed to determine the presence of thesubject fusion proteins.

[0090] Specifically, since the subject fusion kinases of the presentinvention result from a chromosomal deletion event, one can determinethe presence of the subject fusion kinases by screening or assaying forthe presence of one or more particular genetic loci that are locatedbetween NM_(—)030917 and PDGFRα and are not present if the chromosomaldeletion event has occurred. Representative genomic sequences that canbe assayed in these embodiments of the subject methods include sequencesfound in genes that are located between NM_(—)030917 and PDGFRα, likethe genes LNX, RPL21, CHIC2, MORF4 or GSH2. Alternatively, one can alsoassay for sequences at 4q12 that do not code for a gene, where thesequence is specific for 4q12. In yet other embodiments, one may screenfor sequences at the very 3′ end of NM_(—)030917 or the 5′ end ofPDGFRα, including sequences coding for the extracellular domain ofPDGFRα but not of sequences coding for the transmembrane ofintracellular domain of PDGFRα.

[0091] In these embodiments, the presence or absence of the targetsequences, as described above, can be assayed using any convenientprotocol. For example, the detection of particular genetic loci can beachieved by using fluorescence in situ hybridization (FISH). In thistechnique, a fluorescently labeled oligonucleotide is used as a probethat will hybridize with its complementary sequence on the respectivechromosome if that complementary sequence is present, whichhybridization can then be detected by fluorescent microscopy. In thepresent situation, given that the 4q12 locus is present twice in thegenetic material of each cell, hybridization of a probe derived fromthat region should result in two distinct hybridization events and,hence, in two fluorescent signals per normal nucleus, if no chromosomaldeletion has occurred. Representative probes that can be employedinclude, but are not limited to: (a) those derived from genes that arelocated between NM_(—)030917 and PDGFRα, such as the genes LNX, RPL21,CHIC2, MORF4 or GSH2; (b) probes derived from sequences at 4q12 that donot code for a gene; and (3) probes derived from a sequence at the very3′ end of NM_(—)030917 or the 5′ end of PDGFRα, including sequencescoding for the extracellular domain of PDGFRα. If a deletion at the 4q12locus has occurred, only one hybridization signal per nucleus will bedetected. The presence of only one hybridization signal per nucleus in asignificant number (e.g., at least about 100, such as about 200 or more)of hematological cells derived from the bone marrow or from peripheralblood of a patient is strongly indicative of a deletion that can haveled to the formation of the fusion gene and therefore can be employed asa diagnostic marker.

[0092] Screening Assays

[0093] Also provided by the subject invention are screening protocolsand assays for identifying agents that modulate, e.g., inhibit orenhance, activity of the subject fusion proteins. As such, the screeningassays are assays that provide for the identification of agents thatmodulate, e.g., inhibit or enhance, the kinase activity of the subjectfusion proteins.

[0094] The screening methods will typically be assays that provide forqualitative/quantitative measurements of fusion protein kinase activity,e.g., of the ability of the fusion protein to catalyze transfer of aphosphoryl group from a donor to an acceptor. For example, the assaycould be an assay which measures the kinase activity of a fusion proteinof the subject invention in the presence and absence of a candidateinhibitor agent. The screening method may be an in vitro or in vivoformat, where both formats are readily developed by those of skill inthe art. In other words, such assays can be done in vivo or in vitro inmammalian cells, non-mammalian cells, yeast, bacteria, etc.

[0095] A. In vitro Models of Fusion Protein Function

[0096] In vitro models of fusion protein function are provided, wherethe in vitro models may be cell-free models or employ the use of cells.Of particular interest are models of fusion protein kinase activity.

[0097] Cell free-models typically include: a fusion protein polypeptideand a candidate modulatory agent, e.g., competitor or inhibitoragent/molecule, where the models further typically include at least oneof: a donor molecule that includes a phsophoryl group (typically ATP)that is to be transferred to an acceptor molecule and an acceptormolecule that is to receive the phosphoryl group transferred by thedonor.

[0098] The competitor may be any compound that is, or is suspected tobe, a compound capable of specifically inhibiting the fusion protein.Depending on the particular model, one or more of, usually one of, thespecified components may be labeled, where by labeled is meant that thecomponents comprise a detectable moiety, e.g., a fluorescent orradioactive tag, or a member of a signal producing system, e.g. biotinfor binding to an enzyme-streptavidin conjugate in which the enzyme iscapable of converting a substrate to a chromogenic product.

[0099] The above cell free in vitro models may be designed a number ofdifferent ways, where a variety of assay configurations and protocolsmay be employed, as are known in the art. For example, one of thecomponents may be bound to a solid support, and the remaining componentscontacted with the support bound component. The above components of themethod may be combined at substantially the same time or at differenttimes, e.g. soluble fusion protein and a competitor ligand may becombined first, and the resultant mixture subsequently combined withbound acceptor molecule. Following the contact step, the subject methodswill generally, though not necessarily, further include a washing stepto remove unbound components, where such a washing step is generallyemployed when required to remove label that would give rise to abackground signal during detection, such as radioactive or fluorescentlylabeled non-specifically bound components. Following the optionalwashing step, the presence of bound fusion protein will then bedetected.

[0100] In alternative in vitro models, the above components may bepresent in a cell free environment in which the fusion protein exhibitskinase activity in the absence of any inhibitor. The kinase activity isthen monitored in the presence and absence of candidate modulatingagents, where kinase activity may be determined using any convenientassay, e.g., the kinase activity assay described in Proc. Nat'l Acad.Sci. USA (2002) 97:2419-2424.

[0101] Also of interest are in vitro models in which cells are employed.There are numerous cell containing in vitro assays which can be readilyadapted by those of skill in the art for the purposes described herein.For example, the activity of an inhibitor of the subject oncokinasefusion polypeptides can be assessed biochemically using cells expressingthe oncogene. Those cells can be a cancer cell line or they can be acell line generated from the transfection of fusion kinase cDNA. As areflection of its constitutive activity, fusion kinase polypeptideautophosphorylates. If cells are incubated with an inhibitor, the levelof kinase inhibition can be evaluated by immunoprecipitating the fusionkinase polypeptide and subsequently, by performing a Western Blotanalysis in which the blotting antibody is selective for phosphotyrosineresidues. The level of kinase inhibition is reflected in the decrease oftyrosine phosphorylation of the fusion kinase polypeptide.

[0102] In another embodiment, the inhibition of the fusion kinasepolypeptide with an inhibitor leads to cellular responses in therespective primary cancer cells and cancer cell lines or appropriatecell lines derived from the transfection of the fusion kinasepolypeptide cDNA. The cellular responses can include decreasedproliferation, differentiation or apoptosis. The inhibition ofproliferation can be assessed by a multitude of assays, including simplecell counting as well as incorporation of BrdU into DNA followed byELISA. Induction of apoptosis can be evaluated by annexin staining usingflow cytometry, assays for the functional integrity of mitochondria (MTTassays) as well as assays to monitor caspase activation or DNAfragmentation.

[0103] B. In Vivo Models of Fusion Protein Function

[0104] A variety of different in vivo models of fusion protein functionare also provided by the subject invention and may be used in thescreening assays of the subject invention. In vivo models of interestinclude engineered cells that include an expression cassette encodingthe subject fusion proteins. Also of interest in the subject screeningassays are multicellular in vivo models, e.g., the transgenic animalmodels described below.

[0105] The subject nucleic acids can be used to generate transgenic,non-human animals or site-specific gene modifications in cell lines.Transgenic animals may be made through homologous recombination, wherethe normal locus is altered. Alternatively, a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike.

[0106] The modified cells or animals are useful in the study of fusionprotein function and regulation. Specific constructs of interest includeanti-sense, which will block expression, expression of dominant negativemutations, and over-expression of fusion protein genes. Where a sequenceis introduced, the introduced sequence may be either a complete orpartial sequence of a gene that is exogenous to the host animal, e.g., ahuman sequence. A detectable marker, such as lac Z, may be introducedinto the locus, where upregulation of expression will result in aneasily detected change in phenotype.

[0107] One may also provide for expression of the gene or variantsthereof in cells or tissues where it is not normally expressed (e.g.,mammalian, non-mammalian, yeast, bacterial, etc. cells), at levels notnormally present in such cells or tissues, or at abnormal times ofdevelopment. DNA constructs for homologous recombination will compriseat least a portion of the gene native to the species of the host animal,wherein the gene has the desired genetic modification(s), and includesregions of homology to the target locus. DNA constructs for randomintegration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.(1990), Meth. Enzymol. 185:527-537.

[0108] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

[0109] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in in vitro culture.The transgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on fusion protein activity.

[0110] Also of interest are assays involving xenotransplants ofhyperproliferative cells and cell lines into immunocompromised hostanimals, e.g., mice, where cellular, e.g., tumor, growth is identifiedas a readout of the inhibition of the fusion protein by a candidateagent being screened.

[0111] Typical in vivo cancer models for solid tumors involve thegrafting of a piece of primary human tumor or, more frequently, of cellsfrom a human tumor cell line onto immunocompromised mice like nude, SCIDor NOD/SCID mice. The human tumor cells engraft, either subcutaneouslyor in a target organ. If the engraftment takes place subcutaneously,tumor growth can be followed and recorded based on size. In order todemonstrate activity of an anti-cancer compound, the engrafted mice aretreated with the compound and the reduction in size of the tumor isrecorded and/or the prolongation of the survival of the mice isfollowed.

[0112] Assays of interest for fluid or liquid tumors, e.g., leukemias,include the following:

[0113] 1) Subcutaneous injection of a liquid tumor cell line into animmunocompromised host animal, e.g., NOD/SCID mice. Although being aliquid tumor, the tumor cells can form a solid tumor subcutaneously inan immunocompromised host. Once the solid tumor has reached a certainsize, administration of the candidate agent starts and activity of thecandidate agent is measured in reduction of tumor size and survival.

[0114] 2) Injection of a cell line (e.g., BaF3 based) expressing thefusion kinase into a host animal, e.g., into the tail vein of a suitablesyngeneic mouse (Balb c). The injected cells proliferate in the blood ofthe animal, accumulate in the spleen and eventually kill the host animaldue to organ damage. The weight of the spleen and the time of survivalfollowing administration of a given candidate agent serves as anindicator for the success of the therapeutic to affect the cancer.

[0115] 3) Transduction of bone marrow stem cells with a retrovirus thatleads to the expression of a mutant kinase. As a result of theexpression of the mutant kinase in hematopoietic stem cells, the cellsproliferate in an unregulated way which eventually kills the hostanimal, e.g., mouse, in which they are present. A candidate agent thatinhibits the transduced activated kinase and prolongs the survival ofthe host animal is one that exhibits activity against the target fusiononcokinase polypeptide.

[0116] Whether the format is in vivo or in vitro, the model beingemployed is combined with the candidate agent and the effect of thecandidate agent on the model is observed and related to the modulatoryactivity of the agent being tested. For example, for screeninginhibitory agents, the model is combined with the candidate agent in anenvironment in which, in the absence of the candidate agent, kinaseactivity is observed. The conditions may be set up in vitro by combiningthe various required components in an aqueous medium, or the assay maybe carried out in vivo, etc.

[0117] A variety of different candidate agents may be screened by theabove methods. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. A preferred class ofcandidate agents includes those that mimic ATP, the co-substrate for thephosphoryl transfer reaction catalyzed by the fusion proteins. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

[0118] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0119] Agents identified in the above screening assays that inhibitactivity of the subject fusion proteins find use in various methods,where representative methods are described below.

[0120] Methods of Modulating Fusion Protein Activity

[0121] Also provided by the subject invention are methods of modulating,including enhancing and repressing, the activity of the subject fusionproteins. As such, methods of both increasing and decreasing fusionprotein kinase activity are provided. In many embodiments, such methodsare methods of inhibiting fusion protein kinase activity.

[0122] One representative method of inhibiting fusion protein activityis to employ small molecules that inhibit the fusion protein activity.Naturally occurring or synthetic small molecule compounds of interestinclude numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs (especially of ATP) orcombinations thereof. Such molecules may be identified, among otherways, by employing the screening protocols described above.

[0123] In yet other embodiments, expression of the target fusion proteinis inhibited. Inhibition of target fusion protein expression may beaccomplished using any convenient means, including administration of anagent that inhibits target fusion protein expression (e.g., antisenseagents), inactivation of the encoding gene, e.g., through recombinanttechniques, etc.

[0124] Antisense molecules can be used to down-regulate expression ofthe target protein in cells. The anti-sense reagent may be antisenseoligonucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such anti-sense molecules as RNA. The antisense sequence iscomplementary to the mRNA of the targeted gene, and inhibits expressionof the targeted gene products. Antisense molecules inhibit geneexpression through various mechanisms, e.g., by reducing the amount ofmRNA available for translation, through activation of RNAse H, or sterichindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise multiple differentsequences.

[0125] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

[0126] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0127] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993), supra, and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

[0128] Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

[0129] As an alternative to anti-sense inhibitors, catalytic nucleicacid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be usedto inhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International Patent Application WO 95/23225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 95/06764.Conjugates of anti-sense ODN with a met al complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

[0130] In another embodiment, the target protein gene is inactivated sothat it no longer expresses the target fusion protein. By inactivated ismeant that the gene, e.g., coding sequence and/or regulatory elementsthereof, is genetically modified so that it no longer expresses aprotein, or at least a functional protein. The alteration or mutationmay take a number of different forms, e.g., through deletion of one ormore nucleotide residues in the fusion protein region, through exchangeof one or more nucleotide residues in the fusion protein region, and thelike. One means of making such alterations in the coding sequence is byhomologous recombination. Methods for generating targeted genemodifications through homologous recombination are known in the art,including those described in: U.S. Pat. Nos. 6,074,853; 5,998,209;5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744;5,721,367; 5,614,396; 5,612,205; the disclosures of which are hereinincorporated by reference.

[0131] The above-described methods of inhibiting fusion protein activityfind use in a number of different applications. In many applications,the subject methods and compositions are employed to inhibit fusionprotein activity in a cell that endogenously comprises a coding sequencefor the target fusion protein. Expression of the target gene isconsidered to be inhibited if, consistent with the above description,expression is decreased by at least about 2 fold, usually at least about5 fold and often by at least about 25, about 50, about 100 fold or more,as compared to a control, e.g., an otherwise identical cell notsubjected to the subject methods.

[0132] A more specific application in which the subject methods find useis to decrease the proliferative capacity of a cell. The term“proliferative capacity” as used herein refers to the number ofdivisions that a cell can undergo, and preferably to the ability of thetarget cell to continue to divide. The subject methods typically resultin a decrease in proliferative capacity of at least about 1.2-2 fold,usually at least about 5 fold and often at least about 10, 20, 50 foldor even higher, compared to a control.

[0133] Another specific application in which the subject methods finduse is to induce apoptosis, or programmed cell death, in a cell. Thesubject methods typically result in a decrease in the viable cell countsof at least 20%, usually at least 50%, and often of at least 90% or evenhigher.

[0134] Therapeutic Applications of Fusion Protein Activity Modulation

[0135] The methods also find use in a variety of therapeuticapplications in which it is desired to modulate, e.g., increase ordecrease, and typically decrease, fusion protein kinase activity in atarget cell or collection of cells, where the collection of cells may bea whole animal or portion thereof, e.g., tissue, organ, etc. As such,the target cell(s) may be a host animal or portion thereof. In suchmethods, an effective amount of an active agent that modulates fusionprotein activity, e.g., enhances or decreases oncokinase activity asdesired, is administered to the target cell or cells, e.g., bycontacting the cells with the agent, by administering the agent to theanimal, etc. By effective amount is meant a dosage sufficient tomodulate fusion protein activity in the target cell(s), as desired.

[0136] A variety of different types of agents may be employed, includingthe representative types of candidate agents described above, e.g.,small molecule agents, nucleic acid agents, polypeptide agents, etc.

[0137] In certain embodiments, the agents are pyrimidine derivatives asdescribed in U.S. Pat. No. 5,521,184, the disclosure of which is hereinincorporated by reference. In these embodiments, of interest areN-phenyl-2-pyrimidine-amine derivatives of formula (I):

[0138] wherein

[0139] R₁ is 4-pyrazinyl, 1-methyl-1H-pyrrolyl, amino- or amino-loweralkyl-substituted phenyl wherein the amino group in each case is free,alkylated or acylated, 1H-indolyl or 1H-imidazolyl bonded at afive-membered ring carbon atom, or unsubstituted or loweralkyl-substituted pyridyl bonded at a ring carbon atom and unsubstitutedor substituted at the nitrogen atom by oxygen,

[0140] R₂ and R₃ are each independently of the other hydrogen or loweralkyl,

[0141] one or two of the radicals R₄, R₅, R₆, R₇ and R₈ are each nitro,fluoro-substituted lower alkoxy or a radical of formula (II):

—N(R₉)—C(═X)—(Y)_(k)—R₁₀  (II)

[0142] wherein

[0143] R₉ is hydrogen or lower alkyl,

[0144] X is oxo (O), thio (S), imino (NH), N-lower alkyl-imino,hydroximino or O-lower alkyl-hydroximino,

[0145] Y is oxygen or the group NH,

[0146] k is 0 or 1 and

[0147] R₁₀ is an aliphatic radical having at least 5 carbon atoms, or anaromatic, aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic,heterocyclic or heterocyclic-aliphatic radical,

[0148] and the remaining radicals R₄, R₅, R₆, R₇ and R₈ are eachindependently of the others hydrogen, lower alkyl that is unsubstitutedor substituted by free or alkylated amino, piperazinyl, piperidinyl,pyrrolidinyl or by morpholinyl, or lower alkanoyl, trifluoromethyl,free, etherified or esterifed hydroxy, free, alkylated or acylated aminoor free or esterified carboxy,

[0149] and salts of such compounds having at least one salt-forminggroup.

[0150] In these embodiments:

[0151] 1-Methyl-1H-pyrrolyl is preferably 1-methyl-1H-pyrrol-2-yl or1-methyl-1H-pyrrol-3-yl.

[0152] Amino- or amino-lower alkyl-substituted phenyl R₁ wherein theamino group in each case is free, alkylated or acylated, is phenylsubstituted in any desired position (ortho, meta or para) wherein analkylated amino group is preferably mono- or di-lower alkylamino, forexample dimethylamino, and the lower alkyl moiety of amino-lower alkylis preferably linear C₁-C₃ alkyl, such as especially methyl or ethyl.

[0153] 1H-lndolyl bonded at a carbon atom of the five-membered ring is1H-indol-2-yl or 1H-indol-3-yl.

[0154] Unsubstituted or lower alkyl-substituted pyridyl bonded at a ringcarbon atom is lower alkyl-substituted or preferably unsubstituted 2-,or preferably 3- or 4-pyridyl, for example 3-pyridyl,2-methyl-3-pyridyl, 4-methyl-3-pyridyl or 4-pyridyl. Pyridyl substitutedat the nitrogen atom by oxygen is a radical derived from pyridineN-oxide, i.e. N-oxido-pyridyl, e.g. N-oxido-4-pyridyl.

[0155] Fluoro-substituted lower alkoxy is lower alkoxy carrying at leastone, but preferably several, fluoro substituents, especiallytrifluoromethoxy or preferably 1,1,2,2-tetrafluoro-ethoxy.

[0156] When X is oxo, thio, imino, N-lower alkyl-imino, hydroximino orO-lower alkyl-hydroximino, the group C═X is, in the above order, aradical C═O, C═S, C═N—H, C═N-lower alkyl, C═N—OH or CN—O-lower alkyl,respectively. X is preferably oxo.

[0157] k is preferably 0, i.e. the group Y is not present.

[0158] Y, if present, is preferably the group NH.

[0159] The term “lower” within the scope of this text denotes radicalshaving up to and including 7, preferably up to and including 4 carbonatoms.

[0160] Lower alkyl R₁, R₂, R₃ and R₉ is preferably methyl or ethyl.

[0161] An aliphatic radical R₁₀ having at least 5 carbon atomspreferably has not more than 22 carbon atoms, generally not more than 10carbon atoms, and is such a substituted or preferably unsubstitutedaliphatic hydrocarbon radical, that is to say such a substituted orpreferably unsubstituted alkynyl, alkenyl or preferably alkyl radical,such as C₅-C₇ alkyl, for example n-pentyl. An aromatic radical R₁₀ hasup to 20 carbon atoms and is unsubstituted or substituted, for examplein each case unsubstituted or substituted naphthyl, such as especially2-naphthyl, or preferably phenyl, the substituents preferably beingselected from cyano, unsubstituted or hydroxy-, amino- or4-methyl-piperazinyl-substituted lower alkyl, such as especially methyl,trifluoromethyl, free, etherified or esterified hydroxy, free, alkylatedor acylated amino and free or esterified carboxy. In anaromatic-aliphatic radical R₁₀ the aromatic moiety is as defined aboveand the aliphatic moiety is preferably lower alkyl, such as especiallyC₁-C₂ alkyl, which is substituted or preferably unsubstituted, forexample benzyl. A cycloaliphatic radical R₁₀ has especially up to 30,more especially up to 20, and most especially up to 10 carbon atoms, ismono- or poly-cyclic and is substituted or preferably unsubstituted, forexample such a cycloalkyl radical, especially such a 5- or 6-memberedcycloalkyl radical, such as preferably cyclohexyl. In acycloaliphatic-aliphatic radical R₁₀ the cycloaliphatic moiety is asdefined above and the aliphatic moiety is preferably lower alkyl, suchas especially C₁-C₂ alkyl, which is substituted or preferablyunsubstituted. A heterocyclic radical R₁₀ contains especially up to 20carbon atoms and is preferably a saturated or unsaturated monocyclicradical having 5 or 6 ring members and 1-3 hetero atoms which arepreferably selected from nitrogen, oxygen and sulfur, especially, forexample, thienyl or 2-, 3- or 4-pyridyl, or a bi- or tri-cyclic radicalwherein, for example, one or two benzene radicals are annellated (fused)to the mentioned monocyclic radical. In a heterocyclic-aliphatic radicalR₁₀ the heterocyclic moiety is as defined above and the aliphatic moietyis preferably lower alkyl, such as especially C₁-C₂ alkyl, which issubstituted or preferably unsubstituted.

[0162] Etherified hydroxy is preferably lower alkoxy. Esterified hydroxyis preferably hydroxy esterified by an organic carboxylic acid, such asa lower alkanoic acid, or a mineral acid, such as a hydrohalic acid, forexample lower alkanoyloxy or especially halogen, such as iodine, bromineor especially fluorine or chlorine.

[0163] Alkylated amino is, for example, lower alkylamino, such asmethylamino, or di-lower alkylamino, such as dimethylamino. Acylatedamino is, for example, lower alkanoylamino or benzoylamino.

[0164] Esterified carboxy is, for example, lower alkoxycarbonyl, such asmethoxycarbonyl.

[0165] A substituted phenyl radical may carry up to 5 substituents, suchas fluorine, but especially in the case of relatively large substituentsis generally substituted by only from 1 to 3 substituents. Examples ofsubstituted phenyl that may be given special mention are4-chloro-phenyl, pentafluoro-phenyl, 2-carboxy-phenyl, 2-methoxy-phenyl,4-fluorophenyl, 4-cyano-phenyl and 4-methyl-phenyl.

[0166] Salt-forming groups in a compound of formula (I) are groups orradicals having basic or acidic properties. Compounds having at leastone basic group or at least one basic radical, for example a free aminogroup, a pyrazinyl radical or a pyridyl radical, may form acid additionsalts, for example with inorganic acids, such as hydrochloric acid,sulfuric acid or a phosphoric acid, or with suitable organic carboxylicor sulfonic acids, for example aliphatic mono- or di-carboxylic acids,such as trifluoroacetic acid, acetic acid, propionic acid, glycolicacid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid,malic acid, tartaric acid, citric acid or oxalic acid, or amino acidssuch as arginine or lysine, aromatic carboxylic acids, such as benzoicacid, 2-phenoxy-benzoic acid, 2-acetoxybenzoic acid, salicylic acid,4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such asmandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such asnicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such asmethane-, ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonicacids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid.When several basic groups are present mono- or poly-acid addition saltsmay be formed.

[0167] Compounds of formula (I) having acidic groups, for example a freecarboxy group in the radical R₁₀, may form met al or ammonium salts,such as alkali met al or alkaline earth met al salts, for examplesodium, potassium, magnesium or calcium salts, or ammonium salts withammonia or suitable organic amines, such as tertiary monoamines, forexample triethylamine or tri-(2-hydroxyethyl)-amine, or heterocyclicbases, for example N-ethylpiperidine or N,N′-dimethyl-piperazine.

[0168] Compounds of formula (I) having both acidic and basic groups canform internal salts.

[0169] Of particular interest in these embodiments is a pyrimidinederivative described in this patent in which R₁ is 3-pyridyl, R₂, R₃,R₅, R₆, and R₈ are each hydrogen, R₄ is methyl, and R₇ is a group offormula (II) in which R₉ is hydrogen, X is oxo, k is 0, and R₁₀ is4-[(4-methyl-1-piperazinyl)methyl]phenyl. The mesylate salt of thiscompound having the chemical name4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino-phenyl]benzamidemethanesulfonate is now commonly known as imatinib mesylate and soldunder the trademark Gleevec™.

[0170] In yet other embodiments of interest, the agent is not imatinibmesylate.

[0171] Also of interest are phthalazine compounds of formula (III):

[0172] wherein r is 0 to 2, n is 0 to 2; m is 0 to 4; R₁₁ and R₁₂ (i)are in each case a lower alkyl, or (ii) together form a bridge insubformula (III*)

[0173] or (iii) together form a bridge in subformula (III**):

[0174] wherein one or two of the ring members T₁, T₂, T₃, and T₄ arenitrogen, and the remainder are in each case CH; A, B, D, and E are N orCH, wherein not more than 2 of these radicals are N; G is loweralkylene, acyloxy- or hydroxy-lower alkylene, —CH₂—O—, —CH₂—S—,—CH₂—NH—, oxa, thia, or imino; Q is lower alkyl, especially methyl; R isH or lower alkyl; X is imino, oxa, or thia; Y is aryl, pyridyl, or(un)substituted cycloalkyl; and Z is independently mono- ordisubstituted amino, halogen, alkyl, substituted alkyl, hydroxy,etherified or esterified hydroxy, nitro, cyano, carboxy, esterifiedcarboxy, alkanoyl, carbamoyl, N-mono- or N,N-disubstituted carbamoyl,amidino, guanidino, mercapto, sulfo, phenylthio, phenyl-lower alkylthio,alkylphenylthio, phenylsulfinyl, phenyl-lower alkylsulfinyl,alkylphenylsulfinyl, phenylsulfonyl, phenyl-lower alkylsulfonyl, oralkylphenylsulfonyl; and wherein the dashed lines independentlyrepresent optional double bonds; or an N-oxide of said compound with thestipulation that, if Y is pyridyl or unsubstituted cycloalkyl, X isimino, and the remaining radicals are as defined, then G is selectedfrom the group comprising lower alkylene, —CH₂—O—, —CH₂—S—, oxa andthia; or a salt thereof. Such compounds, e.g., PTK787 (also known asVatalanib), are further described in WO 98/35958, U.S. patentapplication Ser. No. 09/859858, and U.S. Pat. No. 6,258,812 B1; thedisclosure of the latter of which is herein incorporated by reference.

[0175] Also of interest in certain embodiments are the protein tyrosinekinase inhibitors of formula (IV):

[0176] in which:

[0177] (i) R¹³ represents a hydrogen atom or a C₁₋₄alkyl group; and R¹⁴represents a group of formula —A¹—NR¹⁷R¹⁸ in which each of R¹⁷ and R¹⁸independently represents a hydrogen atom or a C₁₋₄alkyl group and A¹represents (CH₂)_(m′), (CH₂)_(n′)—A²—(CH₂)_(p′) or (CH₂CH₂O)_(q′)CH₂CH₂in which m′ is an integer of from 2 to 10, each of n′ and p′ is aninteger of from 1 to 6, A² is CH═CH, phenylene, biphenylene,cyclohexylene or piperazinylene and q′ is 1, 2 or 3;

[0178] (ii) R¹³ and R¹⁴ together represent —A³—NR¹⁹—A⁴— in which each ofA³ and A⁴ independently represents (CH₂)_(r′) or (CH₂CH₂O)_(s′)CH₂CH₂ inwhich r′ is an integer of from 2 to 6, s′ is 1, 2 or 3, and R¹⁹represents a hydrogen atom or a C₁₋₄alkyl group;

[0179] (iii) R¹³ and R¹⁴ together with the nitrogen atom to which theyare attached represent a piperidinyl group, which piperidinyl groupbears a substituent of formula —A⁵—R²⁰ at the 4 position, in which A⁵represents C₁₋₄alkylene and R²⁰ represents piperidin-4-yl; or

[0180] (iv) R¹³ and R¹⁴ together with the nitrogen atom to which theyare attached represent a pyrrolidinyl, piperidinyl or morpholino group;and

[0181] R¹⁵ and R¹⁶ each independently represents a hydrogen atom, ahalogen atom, a C₁₋₄alkyl group, a C₁₋₄alkoxy group, a phenyl groupwhich is unsubstituted or substituted by one or two substituentsselected independently from a halogen atom, a C₁₋₄alkyl group and aC₁₋₄alkoxy group, a group of formula R²¹S(O)₂NR²²—, a group of formulaR²³N(R²⁴)S(O)₂—, a group of formula R²⁵C(O)N(R²⁶)— or a group of formulaR²⁷N(R²⁸)C(O)— in which each of R²¹, R²³, R²⁵ and R²⁷ independentlyrepresents a C₁₋₄alkyl group or a phenyl group which is unsubstituted orsubstituted by one or two substituents selected independently from ahalogen atom, a C₁₋₄alkyl group and a C₁₋₄alkoxy group, and each of R²²,R²⁴, R²⁶ and R²⁸ independently represents a hydrogen atom or a C₁₋₄alkylgroup;

[0182] or a pharmaceutically-acceptable salt thereof.

[0183] An inhibitor of formula (IV) of particular interest, identifiedas THRX-165724, is one in which R¹³ and R¹⁴ and the nitrogen to whichthey are attached form a piperazinyl ring and R¹⁵ and R¹⁶ are bothhydrogen. Compounds of formula (IV) are described in U.S. PatentApplication Serial Nos. 60/343,746, 60/343,813, and Ser. No. 10/327,385,the disclosures of which are herein incorporated by reference.

[0184] Another group of compounds of interest in certain embodiments arecompounds of formula (V):

[0185] wherein:

[0186] R²⁹ is selected from the group consisting of —CN, —X, —CX₃, —R³³,—CO₂R³³, —SO₂R³³, —O—C₁₋₈alkyl that is straight or branched chained,—O-phenyl, —O-napthyl, —O-indolyl, and —O-isoquinolinyl, in which X is ahalogen, and R³³ is hydrogen or a C₁₋₈alkyl that is straight or branchedchained,

[0187] R³⁰ and R³² are each independently selected from the groupconsisting of —O—CH₃, —O—CH₂—CH₃, —O—CH₂—CH═CH₂, —O—CH₂—C≡CH,—O(CH₂)—SO₂—R³³, —O—CH₂—CH(R³⁴)CH₂—R³¹ and —O(—CH₂)_(n″)—R³¹, in whichR³⁴ is —OH, —X, or a C₁₋₈alkyl that is straight or branched chained, n″is 2 or 3, and

[0188] R³¹ is selected from the group consisting of; —OH, —O—CH₃,—O—CH₂—CH₃, —NH₂, —N(—CH₃)₂, —NH—CH₂-phenyl, —NH-phenyl, —CN,—C(═NH)—NH₂, —NH—C(═NH)—NH₂, thiazolyl, oxazolyl, pyrrolidinyl,4,4-difluoropiperidinyl, 3,3,-difluoropiperidinyl,3,3-difluoropyrrolidinyl, morpholinyl, piperidinyl, imidazolyl,1,2,3,-triazolyl, methylpiperidinyl, thiomorpholinyl1,1-dioxide-thiomorpholinyl, —O-4-pyridinyl, 1H-tetrazolyl, piperazinyl,and 4-methylpiperazinyl; and pharmaceutically-acceptable isomers, salts,hydrates, solvates, and prodrug derivatives thereof.

[0189] Among compounds of formula (V), of particular interest is thecompound, identified as MLN518 in which R²⁹ is —O-isopropyl, R³⁰ is—O—(CH₂)₃-piperidin-1-yl, and R³² is —OCH₃. Compounds of formula (V) arefurther described in WO 02/16351, which is incorporated herein byreference.

[0190] Yet another group of compounds of interest in certain embodimentsare compounds of formula (VI):

[0191] wherein:

[0192] R³⁵ is selected from the group consisting of hydrogen, halo,alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,—C(O)R⁴⁸, —NR⁴⁶R⁴⁷, —(CH₂)_(r*)R⁴⁹ and —C(O)NR⁴²R⁴³;

[0193] R³⁶ is selected from the group consisting of hydrogen, halo,alkyl, trihalomethyl, hydroxy, alkoxy, cyano, —NR⁴⁶R⁴⁷, —NR⁴⁶C(O)R⁴⁷,—C(O)R⁴⁸, aryl, heteroaryl, and —S(O)₂NR⁴⁶R⁴⁷;

[0194] R³⁷ is selected from the group consisting of hydrogen, halo,alkyl, trihalomethyl, hydroxy, alkoxy, —C(O)R⁴⁸, —NR⁴⁶R⁴⁷, aryl,heteroaryl, —NR⁴⁶S(O)₂R⁴⁷, —S(O)₂NR⁴⁶R⁴⁷, —NR⁴⁶C(O)R⁴⁷, —NR⁴⁶C(O)OR⁴⁷,and —S(O)₂R⁵³, wherein R⁵³ is alkyl, aryl, aralkyl, heteroaryl orheteroaralkyl;

[0195] R³⁸ is selected from the group consisting of hydrogen, halo,alkyl, hydroxy, alkoxy, and —NR⁴⁶R⁴⁷;

[0196] R³⁹ is selected from the group consisting of hydrogen, alkyl and—C(O)R⁴⁰;

[0197] R⁴¹ is selected from the group consisting of hydrogen, alkyl,aryl, heteroaryl, —C(O)R⁵⁰ and —C(O)R⁴⁰;

[0198] R⁴² and R⁴³ are independently selected from the group consistingof hydrogen, alkyl and aryl;

[0199] R⁴⁰ is selected from the group consisting of hydroxy, alkoxy,aryloxy, —N(R⁴⁴)(CH₂)_(n*)R⁴⁵, and —NR⁴⁶R⁴⁷;

[0200] R⁴⁴ is selected from the group consisting of hydrogen and alkyl;

[0201] R⁴⁵ is selected from the group consisting of —NR⁴⁶R⁴⁷, hydroxy,—C(O)R⁴⁸, aryl, heteroaryl, —N⁺(O⁻)R⁴⁶R⁴⁷, —N(OH)R⁴⁶, and —NHC(O)R^(a),wherein R^(a) is unsubstituted alkyl, haloalkyl, or aralkyl;

[0202] R⁴⁶ and R⁴⁷ are independently selected from the group consistingof hydrogen, alkyl, lower alkyl substituted with hydroxyalkylamino,cyanoalkyl, cycloalkyl, aryl, and heteroaryl; or

[0203] R⁴⁶ and R⁴⁷ may combine to form a heterocyclo group;

[0204] R⁴⁸ is selected from the group consisting of hydrogen, hydroxy,alkoxy, and aryloxy;

[0205] R⁴⁹ is selected from the group consisting of hydroxy, —C(O)R⁴⁸,—NR⁴⁶R⁴⁷ and —C(O)NR⁴⁶R⁴⁷;

[0206] R⁵⁰ is selected from the group consisting of alkyl, cycloalkyl,aryl and heteroaryl; and

[0207] n* and r* are independently 1, 2, 3, or 4;

[0208] or a pharmaceutically-acceptable salt thereof.

[0209] A compound of formula (VI) of particular interest, identified asSU11248, is the compound in which R³⁶ is fluoro, R³⁵, R³⁷, and R³⁸ areeach hydrogen, R³⁹ and R⁴¹ are each methyl, and R⁴⁰ is—N(H)(CH₂)₂N(C₂H₅)₂. Compounds of formula (VI) are described in WO01/60814, the disclosure of which is incorporated herein by reference.

[0210] In one embodiment, the agent employed in the methods of thisinvention is selected from the group consisting of:

[0211] or a pharmaceutically-acceptable salt thereof.

[0212] Also of interest are other protein tyrosine kinase inhibitors.Such inhibitors include, but are not limited to, the tyrosine kinaseinhibitors appearing in Appendix A of the priority provisionalapplications having serial Nos. 60/402,330 filed on Aug. 9, 2002 and60/440,491 filed on Jan. 16, 2003; the disclosures of which are hereinincorporated by reference.

[0213] In the subject methods, the active agent(s) may be administeredto the targeted cells using any convenient means capable of resulting inthe desired modulation of fusion protein activity. Thus, the agent canbe incorporated into a variety of formulations for therapeuticadministration. More particularly, the agents of the present inventioncan be formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols. As such,administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intratracheal, etc., administration.

[0214] In pharmaceutical dosage forms, the agents may be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

[0215] For oral preparations, the agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0216] The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

[0217] The agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0218] Furthermore, the agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols; which melt at bodytemperature, yet are solidified at room temperature.

[0219] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

[0220] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0221] Where the agent of interest is imatinib mesylate, the dosageemployed in certain embodiments is substantially less than that which isemployed for the use of imatinib mesylate in the treatment of chronicmyeloid leukemia (CML). By substantially less is meant at least about2-fold, usually at least about 3-fold and more usually at least about4-fold less than the dosages employed in the treatment of CML, wheretypical dosages of imatinib mesylate employed in the subject methods mayrange from about 30 mg/day to about 300 mg/day, usually from about 50mg/day to about 200 mg/day. In yet other embodiments, the dosageemployed is the same as or greater than that employed in the treatmentof CML. The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0222] The invention further provdes a process for producing apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an agent that inhibits the activity of an oncokinase fusionpolypeptide of the invention. The process comprises admixing apharmaceutically acceptable carrier with an agent wherein the agent isidentified by a screening method comprising (1) contacting an oncokinasefusion polypeptide of the invention with a test agent, and determiningthe effect, if any, of the test agent on the activity of the fusionpolypeptide; or (2) contacting a coding sequence for a fusionpolypeptide of the invention with a test agent, and determining theeffect, if any, of the test agent on the expression of the fusionpolypeptide from the coding sequence.

[0223] Where the agent is a polypeptide, polynucleotide, analog ormimetic thereof, e.g. an antisense molecule, it may be introduced intotissues or host cells by any number of routes, including viralinfection, microinjection, or fusion of vesicles. Jet injection may alsobe used for intramuscular administration, as described by Furth et al.(1992), Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells. For nucleic acidtherapeutic agents, a number of different delivery vehicles find use,including viral and non-viral vector systems, as are known in the art.

[0224] Those of skill in the art will readily appreciate that doselevels can vary as a function of the specific compound, the nature ofthe delivery vehicle, and the like. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means.

[0225] The subject methods find use in the treatment of a variety ofdifferent conditions in which the modulation of the subject oncokinasefusion protein activity in the host is desired. By treatment is meantthat at least an amelioration of the symptoms associated with thecondition afflicting the host is achieved, where amelioration is used ina broad sense to refer to at least a reduction in the magnitude of aparameter, e.g. symptom, associated with the condition being treated. Assuch, treatment also includes situations where the pathologicalcondition, or at least symptoms associated therewith, are completelyinhibited, e.g., prevented from happening, or stopped, e.g. terminated,such that the host no longer suffers from the condition, or at least thesymptoms that characterize the condition.

[0226] A variety of hosts are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

[0227] Methods for inhibiting fusion protein activity according to thesubject invention find use in, among other applications, the treatmentof cellular proliferative disease conditions, including neoplasticdisease conditions, i.e., cancers. In such applications, an effectiveamount of an active agent, e.g., an agent that inhibits fusion proteinactivity, is administered to the subject in need thereof. Treatment isused broadly as defined above, e.g., to include at least an ameliorationin one or more of the symptoms of the disease, as well as a completecessation thereof, as well as a reversal and/or complete removal of thedisease condition, e.g., cure.

[0228] There are many disorders associated with a dysregulation ofcellular proliferation, i.e., cellular hyperproliferative disorders. Theconditions of interest include, but are not limited to, the followingconditions.

[0229] The subject methods may be employed in the treatment of a varietyof conditions where there is proliferation and/or migration of smoothmuscle cells, and/or inflammatory cells into the intimal layer of avessel, resulting in restricted blood flow through that vessel, i.e.neointimal occlusive lesions. Occlusive vascular conditions of interestinclude atherosclerosis, graft coronary vascular disease aftertransplantation, vein graft stenosis, peri-anastomatic prosthetic graftstenosis, restenosis after angioplasty or stent placement, and the like.

[0230] Diseases where there is hyperproliferation and tissue remodellingor repair of reproductive tissue, e.g. uterine, testicular and ovariancarcinomas, endometriosis, squamous and glandular epithelial carcinomasof the cervix, etc. are reduced in cell number by administration of thesubject compounds

[0231] Tumors of interest for treatment include carcinomas, e.g. colon,duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic,renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukemia, acute myelogenous leukemias, non-Hodgkin's lymphomas,chronic lymphocytic leukemia, malignant cutaneous T-cells, mycosisfungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis,T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoidlupus erythematosus, lichen planus, etc.; and the like.

[0232] Some cancers of particular interest include breast cancers, whichare primarily adenocarcinoma subtypes. Ductal carcinoma in situ (DCIS)is the most common type of noninvasive breast cancer. In DCIS, themalignant cells have not metastasized through the walls of the ductsinto the fatty tissue of the breast. Infiltrating (or invasive) ductalcarcinoma (IDC) has metastasized through the wall of the duct andinvaded the fatty tissue of the breast. Infiltrating (or invasive)lobular carcinoma (ILC) is similar to IDC, in that it has the potentialto metastasize elsewhere in the body. About 10% to 15% of invasivebreast cancers are invasive lobular carcinomas.

[0233] Also of interest is non-small cell lung carcinoma. Non-small celllung cancer (NSCLC) is made up of three general subtypes of lung cancer.Epidermoid carcinoma (also called squamous cell carcinoma) usuallystarts in one of the larger bronchial tubes and grows relatively slowly.The size of these tumors can range from very small to quite large.Adenocarcinoma starts growing near the outside surface of the lung andmay vary in both size and growth rate. Some slowly growingadenocarcinomas are described as alveolar cell cancer. Large cellcarcinoma starts near the surface of the lung, grows rapidly, and thegrowth is usually fairly large when diagnosed. Other less common formsof lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignantmesothelioma.

[0234] Melanoma is a malignant tumor of melanocytes. Although mostmelanomas arise in the skin, they also may arise from mucosal surfacesor at other sites to which neural crest cells migrate. Melanoma occurspredominantly in adults, and more than half of the cases arise inapparently normal areas of the skin. Prognosis is affected by clinicaland histological factors and by anatomic location of the lesion.Thickness and/or level of invasion of the melanoma, mitotic index, tumorinfiltrating lymphocytes, and ulceration or bleeding at the primary siteaffect the prognosis. Clinical staging is based on whether the tumor hasspread to regional lymph nodes or distant sites. For disease clinicallyconfined to the primary site, the greater the thickness and depth oflocal invasion of the melanoma, the higher the chance of lymph nodemetastases and the worse the prognosis. Melanoma can spread by localextension (through lymphatics) and/or by hematogenous routes to distantsites. Any organ may be involved by metastases, but lungs and liver arecommon sites.

[0235] Other hyperproliferative diseases of interest relate to epidermalhyperproliferation, tissue remodelling and repair. For example, thechronic skin inflammation of psoriasis is associated with hyperplasticepidermal keratinocytes as well as infiltrating mononuclear cells,including CD4+ memory T cells, neutrophils and macrophages.

[0236] The proliferation of immune cells is associated with a number ofautoimmune and lymphoproliferative disorders. Diseases of interestinclude multiple sclerosis, rheumatoid arthritis and insulin dependentdiabetes mellitus. Evidence suggests that abnormalities in apoptosisplay a part in the pathogenesis of systemic lupus erythematosus (SLE).Other lymphoproliferative conditions include the inherited disorder oflymphocyte apoptosis, which is an autoimmune lymphoproliferativesyndrome, as well as a number of leukemias and lymphomas. Symptoms ofallergies to environmental and food agents, as well as inflammatorybowel disease, may also be alleviated by the compounds of the invention.

[0237] One hyperproliferative disorder of particular interest ishypereosinophilic disorders, e.g., hypereosinophilia syndrome or HES.Patients with hypereosinophilic syndrome (HES) present with persistenthigh eosinophilic cell counts. In the course of the disease acutemyeloid eosinophilic leukemia often develops. The underlying cause forHES is not known. The disease is highly lethal because of significantend-organ damage. For a review of this particular disease of interest,see Bain et al., Curr. Opin. Hematol. (2000) 7:21-25.

[0238] Other disorders of particular interest are systemic mast celldisease (SMCD) (see e.g., Pardanani et al., Blood (Jul. 3, 2003) [Epubahead of print]; and acute myeloid leukemia (AML) of the M4E0 subclass.

[0239] Also provided by the subject methods are pharmacogenomictherapeutic methods. In these pharmacogenomic applications or methods, asubject/host/patient is first diagnosed for the presence or absence ofthe subject fusion proteins or coding sequences therefore, e.g., using adiagnostic protocol such as those diagnostic protocols described above.The subject is then treated using a pharmacological protocol, where thesuitability of the protocol for a particular subject/patient isdetermined using the results of the diagnosis step. As such, the subjectinvention provides methods of rational therapeutic protocoldetermination.

[0240] For example, where the diagnosis results in the determinationthat the host suffers from a disease condition characterized by thepresence of the subject fusion proteins, an appropriate pharmacologicaltreatment protocol, e.g., a protocol employing imatinib mesylate is thenemployed to treat the patient. Alternatively, where a patient isdiagnosed as not having the subject fusion protein, or the patient isdiagnosed as having the fusion protein but also include a variant of thefusion protein that is associated with resistance to a particular activeagent (e.g., the T674I mutation) other protocols are then employed.

[0241] Additional Utilities

[0242] The subject polypeptide and nucleic acid compositions find use ina variety of additional applications. Applications in which the subjectpolypeptide and nucleic acid compositions find use include: (a) theidentification of homologs; (b) the identification of expressionregulatory factors; (c) as probes and primers in hybridizationapplications, e.g. PCR; (d) the identification of expression patterns inbiological specimens; etc.

[0243] A. Identification of Homologs

[0244] Homologs are identified by any of a number of methods. A fragmentof the provided cDNA may be used as a hybridization probe against a cDNAlibrary from the target organism of interest, where low stringencyconditions are used. The probe may be a large fragment, or one or moreshort degenerate primers. Nucleic acids having sequence similarity aredetected by hybridization under low stringency conditions, for example,at 50° C. and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate) andremain bound when subjected to washing at 55° C. in 1×SSC (0.15 M sodiumchloride/0.015 M sodium citrate). Sequence identity may be determined byhybridization under stringent conditions, for example, at 50° C. orhigher and 0.1×SSC (15 mM sodium chloride/01.5 mM sodium citrate).Nucleic acids having a region of substantial identity to the providedsequences, e.g. allelic variants, genetically altered versions of thegene, etc., bind to the provided sequences under stringent hybridizationconditions. By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes.

[0245] B. Identification of Expression Regulatory Factors

[0246] Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. (1995), Mol. Med.1:194-205; Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin andRichard-Foy (1995), Eur. J. Biochem. 232:620-626.

[0247] The regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofgene expression, especially in different tissues or stages ofdevelopment, and to identify cis acting sequences and trans-actingfactors that regulate or mediate gene expression. Such transcription ortranslational control regions may be operably linked to a fusion proteingene in order to promote expression of wild type or altered or otherproteins of interest in cultured cells, or in embryonic, fet al or adulttissues, and for gene therapy.

[0248] C. Probes and Primers

[0249] Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i.e. greater than 100 ntare useful for production of the encoded polypeptide, as described inthe previous section. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime extension towards each other.

[0250] D. Identification of Expression Patterns in Biological Specimens

[0251] The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of gene expression in the sample.

[0252] E. Preparation of Mutants

[0253] The sequence of a gene, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two or more, e.g., 5, 10, 20 or morenucleotides or amino acids. The sequence changes may be substitutions,insertions, deletions, or a combination thereof. Deletions may furtherinclude larger changes, such as deletions of a domain or exon. Othermodifications of interest include epitope tagging, e.g. with the FLAGsystem, HA, etc. For studies of subcellular localization, fusionproteins with green fluorescent proteins (GFP) may be used.

[0254] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene37:111-23; Colicelli et al. (1985), mol. Gen. Genet. 199:537-9; andPrentki et al. (1984), Gene 29:303-13. Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al.(1993), Gene 126:3541; Sayers et al. (1992), Biotechniques 13:592-6;Jones and Winistorfer (1992), Biotechniques 12:528-30; Barton et al.(1990), Nucleic Acids Res 18:7349-55; Marotti and Tomich (1989), GeneAnal. Tech. 6:67-70; and Zhu (1989), Anal Biochem 177:120-4. Suchmutated genes may be used to study structure-function relationships, orto alter properties of the protein that affect its function orregulation.

[0255] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0256] Materials and Methods

[0257] A. Compounds

[0258] Imatinib mesylate was extracted from capsules of Gleevec®.Vatalanib was prepared according to the published procedure (Bold etal., J. Med. Chem. (2000) 43:2310-2323. THRX-165724 was prepared bycoupling piperazine to the carboxyl group of SU6668 (Sun et al., J. Med.Chem. (1999) 42:5120-5130) as described in Example 1a of U.S. patentapplication Ser. No. 10/327,385.

[0259] B. cDNA Cloning, Plasmids and Oligos

[0260] Total RNA was isolated from 5×10⁷ EOL-1 cells using the RNeasykit (Qiagen). 100 ng of total RNA was used to make cDNA in a volume of20 μl with a reverse oligonucleotide that primes in the 3′ untranslatedregion of PDGFRα (5′-tccgcattgcaataaagtgg-3′ (SEQ ID NO:13 (from base3478-3459; accession no. M22734)) and the Thermoscript RT-PCR System(Gibco-BRL). 2 μl of the cDNA solution served as template in a 100 μLPCR reaction to amplify full-length NM_(—)030917-PDGFRα (forward oligo:5′-gttgcgctcggggcggccat-3′ (SEQ ID NO:14)(from base 150-169; accessionno. NM_(—)030917), reverse oligo: 5′-ttctgaacgggatccagagg-3′ (SEQ IDNO:15)(from base 3456-3437; accession no. M22734)). The PCR fragment wasisolated by agarose gel electrophoresis, cloned into the TOPO-PCR vector(Invitrogen) and sequenced. The error-free sequence of a splice variantlacking the two observed alternatively spliced exons was cloned into themammalian expression vector pcDNA 3.1(+) (Invitrogen). Patient cDNA wasgenerated as described above, using random hexamers or the specificPDGFRα primers 5′-ggatgtcggaatatttagaa-3′ (SEQ ID NO:16) and5′-gcagaaaggtactgcctttc-3′ (SEQ ID NO:17). To analyze patient cDNA forNM_(—)030917-PDGFRα fusion transcripts the following primer pair wasused: NM_(—)030917 forward: 5′-aattatgggtttaatgaag-3′ (SEQ IDNO:18)(from base 651-699; accession no. NM_(—)030917), PDGFRα reverse:5′-aactttcatgacaggttgg-3′ (SEQ ID NO:19)(from base 2000-1982; accessionno. M22734). For the PCR analysis of the genomic fusion point in EOL-1as well as the two patients, an oligonucleotide priming 3′ of PDGFRαexon 12 in the reverse orientation was combined with specific forwardprimers: PDGFRα genomic reverse: 5′-ttcttactaagcacaagctcagatc-3′ (SEQ IDNO:20)(from base 13912-13888; accession no. AC098587); EOL-1 and patient3 genomic forward: 5′-aagcatctaattaggtgaaactg-3′ (SEQ ID NO:20)(frombase 48554-48576; accession no. NT_(—)022853). Patient 1 genomicforward: 5′-cagggaagaactggaaactc-3′ (SEQ ID NO:22)(from base22466-22485; accession no. NT_(—)022853).

[0261] C. Cells and Cell Lines

[0262] The EOL-1 and the BaF3 cell lines were obtained from the DSMZ(Braunschweig, Germany). The basic culture medium for the EOL-1 and BaF3cell lines was RPMI1640 (Gibco-BRL) supplemented with 10% FBS, 100 U/mlpenicillin and 100 U/ml streptomycin. The medium for BaF3 cells was alsosupplemented with 1 ng/ml IL-3 (Biosource International). A BaF3 cellline expressing NM_(—)030917-PDGFRα was created by electroporation ofBaF3 cells at 300 mV/960 μF. After electroporation, the BaF3 cells weremaintained in IL-3 containing medium for 48 h, selected in IL-3containing medium plus 1 mg/ml G418 for 10 days, and subcloned bylimiting dilution.

[0263] D. Cell Viability Assays

[0264] Cell viability was assessed by tetrazolium salt reduction usingthe MTT assay (Roche). In a 96-well plate, 5×10⁴ cells/well were platedin the presence of serial dilutions of compounds. The cells wereincubated for 72 h prior to addition of MTT substrate.

[0265] E. Immunoprecipitation and Western Blotting

[0266] Antibodies against PDGFRα/β and against phosphotyrosine (4G10)were purchased from Upstate Biotechnology. For each immunoprecipitation,1×10⁷ cells were lysed in 0.75 ml modified RIPA buffer (50 mM Tris-HClpH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, Proteaseinhibitor cocktail (Roche)). The lysates were incubated with theappropriate antibody and Protein G beads (Sigma) overnight at 4° C. Theimmunocomplexes were recovered by centrifugation, washed with RIPAbuffer, boiled in sample buffer and resolved by SDS-PAGE. The proteinswere transferred to a PVDF membrane (Invitrogen), blocked with PBS/0.1%Tween/3% BSA and probed with a specific antibody for 3 h at roomtemperature. Subsequently, the blots were washed with PBS/0.1% Tween.Specific antibody binding was detected with a horseradish-peroxidasecoupled secondary antibody followed by enhanced chemiluminescence ECL(Amersham) and exposure to film. The primary antibody was typicallystripped with ImmunoPure IgG Elution Buffer (Pierce) for re-probing ofthe blot with a second antibody.

[0267] F. Phosphorylation Inhibition Assay

[0268] Cells (1×10⁷) were incubated in 3 ml media with the indicatedconcentration of drug for 1 h. The cells were subsequently lysed andimmunoprecipitated with the appropriate antibody. Then, SDS-PAGE wasperformed followed by immunoblotting with the anti-phosphotyrosineantibody 4G10.

[0269] G. Protein Digestion and Peptide Analysis

[0270] This work was performed by Proteomic Research Services, Inc.(PRS, Ann Arbor, Mich.). The sample was provided to PRS in the form of50 μl of protein G immunoaffinity resin to which was bound tyrosinephosphoproteins from 1×10⁸ EOL-1 cells via antibody 4G10. The proteinswere fractionated by SDS-PAGE and visualized by staining with SYPROruby. Plugs were chosen for excision based on an overlay of theSYPRO-stained lane with that of a companion lane visualized by Westernblotting with 4G10. The plugs were subjected to in-gel digestion withtrypsin (ProGest) and a portion of the supernatant was used for analysisby matrix-assisted laser desorption-ionization mass spectrometry(MALDI/MS). MALDI/MS data were acquired on an Applied Biosystems VoyagerDE-STR instrument and the observed m/z values were submitted to a searchfor peptide mass fingerprints by the software package ProFound fromProteometrics, querying the NCBInr database. In cases where MALDI/MSanalysis was inconclusive, samples were analyzed by nano liquidchromatography followed by 2-dimensional mass spectrometry (LC/MS/MS) ona Micromass Q-Tof2 instrument. The MS/MS data were searched using thesearch engine Mascot from Matrix Science (www.matrixscience.com).

Example 1

[0271] Discovery of a Novel Oncokinase in Hypereosinophilic Syndrome

[0272] Imatinib mesylate (STI-571/Novartis or Gleevec™) (hereinafter“imatinib”) was tested against various CML and AML cell lines.Suprisingly, it was discovered that imatinib was a potent inducer ofapoptosis in EOL-1 cells. The EOL-1 cell line was established in 1984from the peripheral blood of a 33-year-old man with acute myeloid(eosinophilic) leukemia following hypereosinophilic syndrome. The abovediscovery was subsequently confirmed by a report that four HES patientsresponded well to imatinib therapy. See Schaller et al., Med. Gen. Med.(Sep. 7, 2001) 3:9; and Lancet (May 4, 2002); 359(9317):1577-8.

[0273] In a 96-well plate, MV4-11, BV173 and EOL-1 cells were incubatedwith increasing concentrations of imatinib for 72 hours. Then, using theMTT assay, the viability of the cells was assessed. The absorbance at550 nM is a measure for viability. The results are provided graphicallyin FIG. 1.

[0274] As can be seen in FIG. 1, imatinib potently modulates theviability of EOL-1 cells (IC₅₀: ˜100 pM). Specifically, imatinib is avery potent inhibitor of eosinophilic cell line EOL-1. The CML cell lineBV173 also shows reduced viability in the presence of imatinib but thedrug is considerably less potent against this cell line (IC₅₀: 250 nM).Even at 10 μM, imatinib has very little effect on the viability ofMV4-11 cells. The MV4-11 cell line is an acute myeloid (myelomonocytic)leukemia cell line.

[0275] Imatinib was expected to reduce the viability of the CML cellline BV173 since these cells express Bcr-Abl. MV4-11 cells do notexpress Bcr-Abl and as a result they are not sensitive to the drug. Thehigh sensitivity of EOL-1 cells towards imatinib was surprising andunexpected. Like cells from most HES patients, EOL-1 cells are known tohave no chromosomal translocations. Hence, the sensitivity of thesecells towards imatinib cannot be based on the inhibition of Bcr-Abl.Furthermore, imatinib is more than 100-fold more potent against EOL-1than against BV173. Other CML cell lines were tested against imatiniband all of them showed IC₅₀s similar to the one obtained for BV173. Thelack of the typical CML chromosomal translocation and the extraordinarysensitivity of EOL-1 towards imatinib indicated that imatinib wasinhibiting one or more novel targets in these cells.

[0276] In order to identify the novel imatinib target in EOL-1, thephosphoprotein profile in EOL-1 cells was first observed. EOL-1 cellswere left untreated or were treated with increasing concentrations ofimatinib for 2 hours. The cells were subsequently lysed andimmunoprecipitated with an antibody against phosphotyrosine (4G10,obtained from Upstate Biotechnology). Subsequently, a Western Blotanalysis was performed using the same anti-phosphotyrosine antibody.

[0277] The results demonstrated that, in EOL-1, there is a prominentphosphoprotein of 110 kDa molecular weight. This 110 kDa phosphoproteinis not phosphorylated in the presence of imatinib (at about 100 nM).

[0278] The 110 kDa phosphoprotein was predicted to be an activatedkinase that is inhibited by imatinib. To further characterize theprotein, a slice containing the 110 kDa molecular weight region was cutfrom a gel containing immunoprecipitated EOL-1 phosphoproteins. Theproteins in the gel slice were digested with the protease trypsin andthe identities of the tryptic peptide fragments were determined usingmass spectroscopy. Three proteins were identified:

[0279] 1. Nucleolin. Nucleolin is an abundant, 105 kDa phosphoproteininvolved in the assembly of ribosomes. Based on these known features,Nucleolin was determined not to be the likely target for imatinib.

[0280] 2. NM_(—)030917 gene product. This gene is expressed in manytissues but its function is not known. However, it is not a kinase andtherefore is unlikely to be a direct target for imatinib. All theNM_(—)030917 gene product peptides mapped to the N-terminus of theprotein.

[0281] 3. PDGFRα. PDGFRα is a receptor tyrosine kinase. Imatinib isknown to inhibit the closely related PDGFRβ with an IC₅₀ of 300-1000 nM.Therefore, PDGFRα was identified as a potential candidate for theimatinib target in EOL-1. Surprisingly, all the PDGFRα peptidesidentified mapped into the C-terminus of the protein. The C-terminuscontains the kinase domain of the receptor.

[0282] Accordingly, the following experiment was performed to verifythat the 110 kDa phosphoprotein contains the C-terminus of the PDGFRαreceptor.

[0283] EOL-1 cells were left untreated or were treated with increasingconcentrations of imatinib for 2 hours. The cells were lysed and thelysates were immunoprecipitated with an anti-PDGFRα antibody thatrecognizes an epitope in the C-terminus of the receptor. Subsequently, aWestern Blot analysis was performed using the anti-phosphotyrosineantibody.

[0284] It was found that the anti-phosphotyrosine antibodyimmunoprecipitated a phosphoprotein of 110 kDa. This phosphoprotein wasdephosphorylated in the presence of imatinib. The IC₅₀ was ˜30 nM. Thisdata suggested that the 110 kDa phosphoprotein in EOL-1 contains theC-terminus of PDGFRα.

[0285] PDGFRα is a receptor with a molecular weight of 185 kDa in itswild-type form. The fact that the C-terminus of PDGFRα was clearlypresent in the 110 kDa phosphoprotein meant that the receptor wasmutated. Such a mutation could also explain why the kinase domain of thereceptor was constitutively activated. Activated tyrosine kinases play arole in many cancers. Often, the activation is a result of a mutation inthe kinase. There are two principal sets of mechanisms by which tyrosinekinases are found to be activated by mutation. The first mechanismsinclude point mutations, deletions or small duplications in the geneencoding the kinase. The second mechanisms include the formation offusion proteins like Bcr-Abl which are usually a result of chromosomaltranslocation.

[0286] The above findings supported a conclusion that there was a fusionbetween the PDGFRα kinase domain and a second protein, analogous toBcr-Abl. It is known that there are no gross chromosomal abnormalitiesin EOL-1 cells. There is a small deletion on chromosome 9 but the PDGFRαresides on chromosome 4. If PDGFRα was fused to another gene, that genehad to be nearby on chromosome 4 in order not to be cytogeneticallyobvious. NM_(—)030917, the gene product of which was found to be presentin the same gel slice as PDGFRα, is located in close proximity to thePDGFRα gene on chromosome 4. Thus, a small rearrangement on chromosome 4would lead to the fusion of these two genes.

[0287] To test this possibility, RNA from EOL-1 cells was isolated andused to generate cDNA with a primer that primes in the non-coding, 3′region of the PDGFRα gene. The cDNA was used as a template for a PCRreaction with a primer pair priming at the very 3′ end of the PDGFRα andat the very 5′ end of NM_(—)030917. The PCR yielded a fragment of about2.5 kb that was cloned. Sequencing of various clones revealed thefollowing:

[0288] 1. NM_(—)030917 and PDGFRα form a fusion transcript in which theintracellular domain of PDGFRα containing the kinase domain is fused tothe N-terminus of NM_(—)030917.

[0289] 2. The fusion is in frame giving rise to an open reading sequencecomprising 2502 base pairs corresponding to 834 amino acids.

[0290] 3. The NM_(—)030917 fragment is alternatively spliced resultingin the optional addition of one or two exons.

[0291] 4. A tryptic peptide encompassing amino acid sequence from thepredicted NM_(—)030917 and PDGFRα fusion point was identified in theEOL-1 110 kDa phosphoprotein gel slice. Hence, the fusion protein isexpressed in EOL-1 cells.

[0292] The coding sequence for the NM_(—)030917-PDGFRα fusion gene is asfollows: ATGTCGGCCGGCGAGGTCGAGCGCCTAGTGTCGGAGCTGAGCGGCGGGACC (SEQ IDNO:05) GGAGGGGATGAGGAGGAAGAGTGGCTCTATGGCGGCCCATGGGACGTGCATGTGCACAGTGATTTGGCAAAGGACCTAGATGAAAATGAAGTTGAAAGGCCAGAAGAAGAAAATGCCAGTGCTAATCCTCCATCTGGAATTGAAGATGAAACTGCTGAAAATGGTGTACCAAAACCGAAAGTGACTGAGACCGAAGATGATAGTGATAGTGACAGCGATGATGATGAAGATGATGTTCATGTCACTATAGGAGACATTAAAACGGGAGCACCACAGTATGGGAGTTATGGTACAGCACCTGTAAATCTTAACATCAAGACAGGGGGAAGAGTTTATGGAACTACAGGGACAAAAGTCAAAGGAGTAGACCTTGATGCACCTGGAAGCATTAATGGAGTTCCACTCTTAGAGGTAGATTTGGATTCTTTTGAAGATAAACCATGGCGTAAACCTGGTGCTGATCTTTCTGATTATTTTAATTATGGGTTTAATGAAGATACCTGGAAAGCTTACTGTGAAAAACAAAAGAGGATACGAATGGGACTTGAAGTTATACCAGTAACCTCTACTACAAATAAAATTACGGCCGAAGACTGTACTATGGAAGTTACACCAGGTGCAGAGATCCAAGATGGCAGATTCAATCTTTTTAAGGTACAGCAGGGAAGAACTGGAAACTCAGAGAAAGAAACTGCCCTTCCATCTACAAAAGCTGAGTTTACTTCTCCTCCTTCTTTGTTCAAGACTGGGCTTCCACCGAGCAGAAACAGCACUCTTCTCAGTCTCAGACAAGTACTGCCTCCAGAAAAGCCTCAAGCGTTGGGAAGTGGCAGGATCGATATGGGAGGGCCGAATCACCTGATCTAAGGAGATTACCTGGGGCAATTGATGTTATCGGTCAGACTATAACTATCAGCCGAGTAGAAGGCAGGCGACGGGCAAATGAGAACAGCAACATACAGGACTCAAGATGGGAGTttCCAAGAGATGGACTAGTGCTTGGTCGGGTCTTGGGGTCTGGAGCGTTTGGGAAGGTGGTTGAAGGAACAGCCTATGGATTAAGCCGGTCCCAACCTGTCATGAAAGTTGCAGTGAAGATGCTAAAACCCACGGCCAGATCCAGTGAAAAACAAGCTCTCATGTCTGAACTGAAGATAATGACTCACCTGGGGCCACATTTGAACATTGTAAACTTGCTGGGAGCCTGCACCAAGTCAGGCCCCATTTACATCATCACAGAGTATTGCTTCTATGGAGATTTGGTCAACTATTTGCATAAGAATAGGGATAGCTTCCTGAGCCACCACCCAGAGAAGCCAAAGAAAGAGCTGGATATCTTTGGATTGAACCCTGCTGATGAAAGCACACGGAGCTATGTTATTTTATCTTTTGAAAACAATGGTGACTACATGGACATGAAGCAGGCTGATACTACACAGTATGTCCCCATGCTAGAAAGGAAAGAGGTTTCTAAATATTCCGACATCCAGAGATCACTCTATGATCGTCCAGCCTCATATAAGAAGAAATCTATGTTAGACTCAGAAGTCAAAAACCTCCTTTCAGATGATAACTCAGAAGGCCTTACTTTATTGGATTTGTTGAGCTTCACCTATCAAGTTGCCCGAGGAATGGAGTTTTTGGCTTCAAAAAATTGTGTCCACCGTGATCTGGCTGCTCGCAACGTTCTCCTGGCACAAGGAAAAATTGTGAAGATCTGTGACTTTGGCCTGGCCAGAGACATCATGCATGATTCGAACTATGTGTCGAAAGGCAGTACCTTTCTGCCCGTGAAGTGGATGGCTCCTGAGAGCATCTTTGACAACGTCTACACCACACTGAGTGATGTCTGGTCTTATGGCATTCTGCTCTGGGAGATCTTTTCCCTTGGTGGCACCCCTTACCCCGGCATGATGGTGGATTCTACTTTCTACAATAAGATCAAGAGTGGGTACCGGATGGCCAAGCCTGACCACGCTACCAGTGAAGTCTACGAGATCATGGTGAAATGCTGGAACAGTGAGCCGGAGAAGAGACCCTCCTTTTACCACCTGAGTGAGATTGTGGAGAATCTGCTGCCTGGACAATATAAAAAGAGTTATGAAAAAATTCACCTGGACTTCCTGAAGAGTGACCATCCTGCTGTGGCACGCATGCGTGTGGACTCAGACAATGCATACATTGGTGTCACCTACAAAAACGAGGAAGACAAGCTGAAGGACTGGGAGGGTGGTCTGGATGAGCAGAGACTGAGCGCTGACAGTGGCTACATCATTCCTCTGCCTGACATTGACCCTGTCCCTGAGGAGGAGGACCTGGGCAAGAGGAACAGACACAGCTCGCAGACCTCTGAAGAGAGTGCCATTGAGACGGGTTCCAGCAGTTCCACCTTCATCAAGAGAGAGGACGAGACCATTGAAGACATCGACATGATGGACGACATCGGCATAGACTCTTCAGACCTGGTGG AAGACAGCTTCCTGTAA

[0293] The underlined sequences are alternatively spliced. None or oneor both sequences may be present in a given transcript. These additionalalternatives are provided as SEQ ID NOS: 06, 07 and 08 in the attachedSequence Listing.

[0294] The above coding sequence encodes a NM_(—)030917-PDGFRα fusionprotein product having the following amino acid sequence:MSAGEVERLVSELSGGTGGDEEEEWLYGGPWDVHVHSDLAKDLDENEVERPEEE (SEQ ID NO:01)NASANPPSGIEDETAENGVPKPKVTETEDDSDSDSDDDEDDVHVTIGDIKTGAPQYGSYGTAPVNLNIKTGGRWGTTGTKVKGVDLDAPGSINGVPLLEVDLDSFEDKPWRKPGADLSDYFNYGFNEDTWKAYCEKQKRIRMGLEVIPVTSTTNKITAEDCTMEVTPGAEIQDGRFNLFKVQQGRTGNSEKETALPSTKAEFTSPPSLFKTGLPPSRNSTSSQSQTSTASRKANSSVGKWQDRYGRAESPDLRRLPGAIDVIGQTITISRVEGRRRANENSNIQLPYDSRWEFPRDGLVLGRVLGSGAFGKWEGTAYGLSRSQPVMKVAVKMLKPTARSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIITEYCFYGDLVNYLHKNRDSFLSHHPEKPKKELDIFGLNPADESTRSYVILSFENNGDYMDMKQADTTQYVPMLERKEVSKYSDIQRSLYDRPASYKKKSMLDSEVKNLLSDDNSEGLTLLDLLSFTYQVARGMEFLASKNCVHRDLAARNVLLAQGKIVKICDFGLARDIMHDSNYVSKGSTFLPVKWMAPESIFDNLYTTLSDVWSYGILLWEIFSLGGTPYPGMMVDSTFYNKIKSGYRMAKPDHATSEVYEIMVKCWNSEPEKRPSFYHLSEIVENLLPGQYKKSYEKIHLDFLKSDHPAVARMRVDSDNAYIGVTYKNEEDKLKDWEGGLDEQRLSADSGYIIPLPDIDPVPEEEDLGKRNRHSSQTSEESAIETGSSSSTFIKREDETIEDIDMMDDIGIDSSDLV EDSFL*

[0295] The underlined peptides are alternatively spliced sequences. Noneor one or both sequences may be present in a given fusion protein. Thesealternative sequences are provided as SEQ ID NOS: 02, 03 and 04 in theattached sequence listing.

[0296] Double Underline: Peptide identified by mass spec which containssequences from NM_(—)030917 and PDGFRα. (The fusion is between Q(NM_(—)030917) and L (PDGFRα)

[0297] Primer pairs flanking the fused exons of NM_(—)030917 and PDGFRαon the normal chromosome 4 were designed. Using genomic DNA, those exonsin EOL-1 cells as well as other leukemia cell lines were amplified usingthe designed primer pairs in a PCR protocol. When the 5′ primer of thefused NM_(—)030917 exon was combined with the 3′ primer of the fusedPDGFRα exon, a fragment of 1100 base pairs from EOL-1 genomic DNA butnot from any other cell line tested was obtained in the PCR protocol.This fragment contained the genomic recombination point and it isderived from the mutant chromosome 4. The 1100 base pair fragment wassequenced to characterize the genomic recombination point. The followingobservations were made:

[0298] 1. The recombination point deletes approximately one million basepairs on chromosome 4. This leads to the fusion of an intron inNM_(—)030917 to the middle of exon 12 in PDGFRα.

[0299] 2. The splice donor dinucleotide GT in the NM_(—)030917 intronrecognizes the first AG dinucleotide in the PDGFR alpha exon as thesplice acceptor site. The splicing reaction results in a fusion thatmaintains the reading frame of the PDGFR gene.

[0300] Deletion of 1 Megabase fuses NM_(—)030917 and exon 12 of PDGFRαon chromosome 4:

(SEQ ID NO:09)

GTTTAGTATT TTAAAATAAATAATTTTCTTTAACTGAAGGATGATATCAACATTATAATTTAATTTATTCAATAGATAGTCATAGTGCTATGCTGTGTTTTAGGTGATACAAAGAGATTTAAAATGTGAAGCCAGTCATTTATTATAAAGGCATTTCCAGTTAGAATTTATAATCTCTGAATCTTTTTTTTAATAGGGTTTTCTTTCTTTTCCTTGGTAGAAACATAGTACAGGTTGAGTATCTCTTATCTGAAATGCTTGGGACCAGAAGTGTATCAGATTTTGAAATGTTTGGATTTTGAAATGTTCATACGTATGTAATCTTAGAGATGAGACCCAAGTCTAAACATGAAATTTATGTTTCATAAATAAGTTATTTAATTTGTTATAATGATTTGTTAATTTATGATAATCTTTTTTCCTTTGGCAACCCTGAGTAAACTGTGTAGTGGGCCTGAGTTTTGATTGTGATCCGTCACATGTGAGGTTGGGTGTGGAATTTTCCACTTGTGATAAAATGTTGATGCTCAAAATGTTTCGGATTTTAGAGCATTTCAGATTTCAGATTTTTGGATTAGGAATACTCAACTTGTATATAGGTTTACTGAATGAAAAATAAAACACCACCAGTGATTTTACCACCTAAAGATAACCACATCTGGTACATCTCTTGATACTAAGCAAAATTGGGATACTATTATAATTATATTTCTGTGTATACTTTTTTCCCCATCTAAAATTATCATTTGTATGTTTTCATATTCTTAAAATATGATTAAAATATCTTCATGATAGCTTCATTGGATAAATATACCATGATTTATFUAATGTGACATTTCTGGAGGTGGCGTAGAGCTGCTTTGTTTTTAGGTGAAAAATTGAGGGGAGATAAATTATCAGTAAGTTGGATAATTAATAATTAGAACTTTAATAACTGAGACTTCCAGCTATTCATTTTGGCCATACTTTTTTTCATTATTTATTCC . . . about 1 Megabase . . .TGGTTTGAGAGATGGTACTGCCTATCCCTAAAATGAACCAGGCAGCCCTCACAC (SEQ ID NO:10)TTCCCCACCAGCAGTGAGAGATTCCTGGCTCAGACACAGCCACACTACCTTGCTGCCCCTGTGCATGTCTGCCAGGAAACTTTTCATTGTGCCTCTCTCTCTTGTCACGTAGCCCTGCGTTCTGAACTCACGGTGGCTGCTGCAGTCCTGGTGCTGTTGGTGATTGTGATCATCTCACTTATTGTCCTGGTTGTCATTTGGAAACAGGTAGATATTTTCTCATAAAACTAAAGATCTTTGAAGCCAATGAGAACAAGCATAGCAACCTAGTTCAGTGCTTGGCACAGAGAAGGAGCTCAGCAATTACATGTGGAGTGAACGTTGTTGGACTCTACTGTGTCCAGTCACTGTGCTGCTTCAGTGAAGCTCTGGTGCACTGGGACTTTGGTAATTCACCAGTTACCTGTCCTGGTCATTTATAGAAACCGAGGTATGAAATTCGCTGGAGGGTCATTGAATCAATCAGCCCAGATGGACATGAATA

TATTTATGTGGACCCG

GTAAGTTCCATGGGGTAACCTCCCAAGACTCCCTTTTCCCTTGCACACAACTTTACAATTTATAGGCCTTGGCAGAATAGAGATCTGAGCTGTGCTTAGTAAGAACTAGGCAATGGAAATTTGCTTTCAGAAATACATTTCTGT CTTGACAGTAAGTTAANon bold: intron Bold: exon

[0301] Non bold: intron

[0302] Bold: exon italic bases denote the contiguous sequence after the1 megabase deletion has occured.

[0303] ATACAGGTTTAG . . . GACCCGATGCAGCTGCCT (SEQ ID NO:11 and 12)

[0304] The underlined bases denote the splice donor and splice acceptorsites of the intron that comprise the fusion point.

[0305] In summary, eosinophilia cells in patients have been shown toundergo apoptosis when exposed to imatinib. The eosinophilic AML cellline EOL-1 has been shown above to be similarly sensitive to imatiniband therefore represents a good model system to identify targets forimatinib in this disease. The EOL-1 cells were discovered to have achromosomal rearrangement on chromosome 4 which leads to the expressionof a fusion protein consisting of an uncharacterized protein and thecytoplasmic domain of PDGFRα. The fusion protein is highlyphosphorylated in EOL-1 cells which is a reflection of an activatedstate of the PDGFRα kinase domain. The phosphorylation of the fusionprotein can be inhibited with imatinib. The IC₅₀ is ˜30 nM. The abovedata show that the kinase domain of PDGFRα expressed as a fusion proteinis a target for imatinib in EOL-1.

[0306] Thus, the above results show that PDGFRα fusion proteins play animportant role in hypereosinophilic syndrome and acute myeloid(eosinophilic) leukemia, analogous to Bcr-Abl in CML. The fusionproteins may differ in the exact genomic location where NM_(—)030917 andPDGFRα are recombined.

[0307] Furthermore, since fusion with NM_(—)030917 is able to activatethe kinase domain of PDGFRα, then fusion of an N-terminal domain of theNM_(—)030917 protein activates other kinase domains in certainembodiments. For example, just downstream from PDGFRα on chromosome 4are two more receptor tyrosine kinases, namely c-kit and VEGFR-2. Assuch, in other hyperproliferative diseases, fusions of NM_(—)030917 andc-kit or VEGFR-2 are expected to be present.

Example 2

[0308] Inhibition of Cellular NM 030917-PDGFRα Autophosphorylation

[0309] Using Western Blot analysis, the potency of THRX-165724(described in Example 1 of patent application Ser. No.10/327,385) andvatalanib (PTK787) (described in Examples 1 to 4 of U.S. Pat. No.6,258,812 B1) in inhibiting cellular NM_(—)030917-PDGFRαautophosphorylation was assessed. It was found that both of these kinaseinhibitors have activity in this assay. In parallel, both inhibitorswere tested for their ability to induce apoptosis in EOL-1 cells. TheIC₅₀s obtained in the two assays are listed below: Inhibition ofInhibition of Compound Autophosphorylation Cell Viability THRX-16572410-30 nM  10 nM vatalanib (PTK787) 100-300 nM 100 nM

[0310] The IC₅₀s for THRX-165724 and vatalanib (PTK787) correlate wellbetween the two assays. The above results demonstrate that THRX-165724and vatalanib (PTK787) at 10-30 and 100-300 nM, respectively, induceapoptosis by inhibiting NM_(—)030917-PDGFRα.

Example 3

[0311] Cell-Transforming Potential of NM 030917-PDGFRα

[0312] Mutationally activated tyrosine kinases, as found in manycancers, can transform the murine myeloid cell line BaF3 tointerleukin-3 independence. In order to determine if NM_(—)030917-PDGFRαhas the ability to transform cells, a BaF3 cell line was established.BaF3 cells expressing NM_(—)030917-PDGFRα from EOL-1 were found to beIL-3 independent. The fusion protein in these cells was constitutivelyphosphorylated and the phosphorylation was inhibited by imatinib with anIC₅₀ of 30 nM, the same value as obtained in EOL-1. Inhibition withimatinib, vatalanib and THRX-165724 resulted in reduced viability of theBaF3 NM_(—)030917-PDGFRα cells with IC₅₀s similar to the potency of thedrugs in EOL-1. The effect of the inhibitors was overcome in thepresence of IL-3. NM_(—)030917-PDGFRα is also likely to be the targetfor imatinib, vatalanib and THRX-165724 in EOL-1 since the expression ofthe fusion gene conferred IL-3 independent growth to BaF3 cells whichwas inhibited by the three drugs at concentrations similar to those atwhich they inhibited the viability of EOL-1 cells. The viability ofthese BaF3 cells in the presence of the PDGFRα inhibitors could bemaintained by exogenous IL-3.

Example 4

[0313] Identification of NM 030917-PDGFRα in HES Patient Cells

[0314] In order to determine if the NM_(—)030917-PDGFRα fusion waspresent in HES patients, blood cells from four patients diagnosed withHES were obtained. Patients 1 and 2 had been treated with imatinib.Patient 1 responded to treatment, but patient 2 did not. After showing acomplete hematologic remission, patient 1 relapsed and died. Thispatient had multiple clonal cytogenetic abnormalities which led to thediagnosis of CEL. Genomic DNA as well as total RNA and cDNA wereprepared from all patient cells except for patient 1 for whom onlygenomic DNA, but no RNA and cDNA were obtained from cells beforeimatinib treatment. The cDNA samples were subjected to PCR with a primerpair spanning the fusion point determined in EOL-1 cells. In the samplesfrom patients 1 and 3, fragments could be amplified from the cDNA thatconstituted in-frame fusion transcripts between NM_(—)030917 and PDGFRα(FIG. 3A). No NM_(—)030917-PDGFRα fusion was detected in patients 2 and4. In patient 1, the fusion transcript connects exon 8 of NM_(—)030917within exon 12 of PDGFRα. A similar approach as for EOL-1 was used toidentify the genomic breakpoint. In patient 1, the intronic break is atan AG dinucleotide that serves as the splice acceptor site so that exon8 in NM_(—)030917 and part of exon 12 in PDGFRα are fused in-frame inthe fusion transcript (FIG. 3B). The NM_(—)030917-PDGFRα fusion inpatient 1 was detected in genomic DNA preparations derived from cellstaken before the start of imatinib therapy and at the time of relapse.The analysis of NM_(—)030917-PDGFRα cDNA at the time of relapse revealeda point mutation in the PDGFRα kinase domain. The mutation affects aminoacid position 674 in PDGFRα resulting in the substitution of threonineby isoleucine (T674I).

[0315] In patient 3, the fusion transcript as well as the genomic breakare identical to the mutation found in EOL-1 cells (FIGS. 3A and 3B).

[0316] Amplification and sequencing of genomic DNA from EOL-1 cellsrevealed that the genomic breakpoint junctions fell within an intronfollowing exon 11 of NM_(—)030917 and within exon 12 of PDGFRα. The samemutation was observed in patient 3 and a similar submicroscopic deletionwas discovered in patient 1. In patient 1 the resulting transcript fuseda different site in the NM_(—)030917 gene to a distinct site in PDGFRαexon 12. Exon 12 encompasses the cytoplasmic juxtamembrane region ofPDGFRα, followed by the kinase domain.

[0317] Patient 1 relapsed and died after initially having shown acomplete remission in response to imatinib. At the time of relapse, thispatient had a T674I mutation in the PDGFRα kinase domain. T674 in PDGFRαcorresponds to T315 in c-Abl. Based on the crystal structure of thecatalytic domain of c-Abl bound to a derivative of imatinib, T315 formspart of the imatinib binding pocket and establishes a hydrogen bond withthe drug (Schindler et al., Science (2000) 289:1938-1942). The T315Imutation ablates the kinase inhibitory activity of imatinib for Bcr-Abland it is one of the most common mutations found in CML patients who areresistant to the drug (Gorre et al., Science (2001) 293:876-880;Branford et al., Blood (2002) 99:3472-3475). The above indicates thatthat the T674I mutation underlies the relapse of patient 1 and thereforeprovides further evidence that NM_(—)030917-PDGFRα is the target ofimatinib.

[0318] Two reports describe patients with myeloproliferative disorderswith eosinophilia who have chromosomal translocations at 4q11-12 (Duellet al., Cancer Genet. Cytogenet. (1997) 94:91-94; Schoffski et al., Ann.Hematol. (2000) 79:95-98). These translocations may involve eitherNM_(—)030917 or PDGFRα leading to different disease promoting fusionproteins. For example, NM_(—)030917 may play a role analogous to thatplayed by Tel or Bcr-promoting dimerization and, thus, the activation ofknown oncogenic fusion kinases. Like PDGFRα, the c-Kit gene is locatedon chromosome 4q12. Submicroscopic deletions could also result inNM_(—)030917-c-Kit fusions. A search of the NCBI EST database revealsthat NM_(—)030917 is expressed in many tissues and organs suggestingthat NM_(—)030917-fusion kinases may not be restricted to cells ofhematological origin. The protein encoded by NM_(—)030917 is homologous(26% over 307 residues) to yeast protein FIP1, a component of apolyadenylation factor (Preker et al., Cell (1995) 81:379-389).

[0319] PDGFRα shares a high degree of homology with PDGFRβ (Matsui etal., Science (1989) 243: 800-804. PDGFRβ is a target for variouschromosomal translocations in chronic myeloproliferative diseases thatresult in the expression of fusion kinases; e.g., Tel-PDGFRβ andRab5-PDGFRβ (Golub et al., Cell (1994) 77:307-316; Magnusson et al.,Blood (2001) 98:2518-2525; Ross et al., Blood (1998) 91:44194426; Abe etal., Blood (1997) 90:4271-4277; Kulkarni et al., Cancer Res. (2000) 60:3592-3598; Schwaller et al., Blood (2001) 97:3910-3918. Like PDGFRβ,PDGFRα has recently been described as part of a fusion gene. Baxter etal. (Hum. Mol. Genet. (2002) 11:1391-1397) identified two atypical CMLpatients with a t(4;22)(q12;q11) translocation resulting in Bcr-PDGFRαfusions. Similar to the NM_(—)030917-PDGFRα fusion we have discovered,both Bcr-PDGFRα fusions involve translocation into exon 12 of PDGFRα.Indeed, one of the two Bcr-PDGFRα fusions produces precisely the samePDGFRα fragment that we observed in NM_(—)030917-PDGFRα from EOL-1cells.

[0320] In summary, the above experiments demonstrate that the novelNM_(—)030917-PDGFRα genomic rearrangement, discovered in theeosinophilic EOL-1 cell line, is present in a subset of patientsdiagnosed with HES. Cell viability and phosphorylation data show thatthe NM_(—)030917-PDGFRα kinase encoded by the novel fusion gene plays acentral role in the disease process of these HES patients. Accordingly,HES in which the NM_(—)030917-PDGFRα fusion is detected may be describedas chronic eosinophilic leukemia.

[0321] It is evident from the above results and discussion that thesubject invention provides for important new targets for the treatmentof various disease conditions, including proliferative diseases, such ascancer. In addition to providing the subject targets, the inventionfurther provides important new methods of diagnosis and treatment, whichwill provide significant benefits the medical and related fields.Accordingly, the subject invention represents a significant contributionto the art.

[0322] All publications and patents cited in this specification areherein incorporated by reference as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. The citation of any publication is for its disclosureprior to the filing date and should not be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention.

[0323] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1 22 1 849 PRT homo sapiens 1 Met Ser Ala Gly Glu Val Glu Arg Leu ValSer Glu Leu Ser Gly Gly 1 5 10 15 Thr Gly Gly Asp Glu Glu Glu Glu TrpLeu Tyr Gly Gly Pro Trp Asp 20 25 30 Val His Val His Ser Asp Leu Ala LysAsp Leu Asp Glu Asn Glu Val 35 40 45 Glu Arg Pro Glu Glu Glu Asn Ala SerAla Asn Pro Pro Ser Gly Ile 50 55 60 Glu Asp Glu Thr Ala Glu Asn Gly ValPro Lys Pro Lys Val Thr Glu 65 70 75 80 Thr Glu Asp Asp Ser Asp Ser AspSer Asp Asp Asp Glu Asp Asp Val 85 90 95 His Val Thr Ile Gly Asp Ile LysThr Gly Ala Pro Gln Tyr Gly Ser 100 105 110 Tyr Gly Thr Ala Pro Val AsnLeu Asn Ile Lys Thr Gly Gly Arg Val 115 120 125 Tyr Gly Thr Thr Gly ThrLys Val Lys Gly Val Asp Leu Asp Ala Pro 130 135 140 Gly Ser Ile Asn GlyVal Pro Leu Leu Glu Val Asp Leu Asp Ser Phe 145 150 155 160 Glu Asp LysPro Trp Arg Lys Pro Gly Ala Asp Leu Ser Asp Tyr Phe 165 170 175 Asn TyrGly Phe Asn Glu Asp Thr Trp Lys Ala Tyr Cys Glu Lys Gln 180 185 190 LysArg Ile Arg Met Gly Leu Glu Val Ile Pro Val Thr Ser Thr Thr 195 200 205Asn Lys Ile Thr Ala Glu Asp Cys Thr Met Glu Val Thr Pro Gly Ala 210 215220 Glu Ile Gln Asp Gly Arg Phe Asn Leu Phe Lys Val Gln Gln Gly Arg 225230 235 240 Thr Gly Asn Ser Glu Lys Glu Thr Ala Leu Pro Ser Thr Lys AlaGlu 245 250 255 Phe Thr Ser Pro Pro Ser Leu Phe Lys Thr Gly Leu Pro ProSer Arg 260 265 270 Asn Ser Thr Ser Ser Gln Ser Gln Thr Ser Thr Ala SerArg Lys Ala 275 280 285 Asn Ser Ser Val Gly Lys Trp Gln Asp Arg Tyr GlyArg Ala Glu Ser 290 295 300 Pro Asp Leu Arg Arg Leu Pro Gly Ala Ile AspVal Ile Gly Gln Thr 305 310 315 320 Ile Thr Ile Ser Arg Val Glu Gly ArgArg Arg Ala Asn Glu Asn Ser 325 330 335 Asn Ile Gln Leu Pro Tyr Asp SerArg Trp Glu Phe Pro Arg Asp Gly 340 345 350 Leu Val Leu Gly Arg Val LeuGly Ser Gly Ala Phe Gly Lys Val Val 355 360 365 Glu Gly Thr Ala Tyr GlyLeu Ser Arg Ser Gln Pro Val Met Lys Val 370 375 380 Ala Val Lys Met LeuLys Pro Thr Ala Arg Ser Ser Glu Lys Gln Ala 385 390 395 400 Leu Met SerGlu Leu Lys Ile Met Thr His Leu Gly Pro His Leu Asn 405 410 415 Ile ValAsn Leu Leu Gly Ala Cys Thr Lys Ser Gly Pro Ile Tyr Ile 420 425 430 IleThr Glu Tyr Cys Phe Tyr Gly Asp Leu Val Asn Tyr Leu His Lys 435 440 445Asn Arg Asp Ser Phe Leu Ser His His Pro Glu Lys Pro Lys Lys Glu 450 455460 Leu Asp Ile Phe Gly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr 465470 475 480 Val Ile Leu Ser Phe Glu Asn Asn Gly Asp Tyr Met Asp Met LysGln 485 490 495 Ala Asp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys GluVal Ser 500 505 510 Lys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp Arg ProAla Ser Tyr 515 520 525 Lys Lys Lys Ser Met Leu Asp Ser Glu Val Lys AsnLeu Leu Ser Asp 530 535 540 Asp Asn Ser Glu Gly Leu Thr Leu Leu Asp LeuLeu Ser Phe Thr Tyr 545 550 555 560 Gln Val Ala Arg Gly Met Glu Phe LeuAla Ser Lys Asn Cys Val His 565 570 575 Arg Asp Leu Ala Ala Arg Asn ValLeu Leu Ala Gln Gly Lys Ile Val 580 585 590 Lys Ile Cys Asp Phe Gly LeuAla Arg Asp Ile Met His Asp Ser Asn 595 600 605 Tyr Val Ser Lys Gly SerThr Phe Leu Pro Val Lys Trp Met Ala Pro 610 615 620 Glu Ser Ile Phe AspAsn Leu Tyr Thr Thr Leu Ser Asp Val Trp Ser 625 630 635 640 Tyr Gly IleLeu Leu Trp Glu Ile Phe Ser Leu Gly Gly Thr Pro Tyr 645 650 655 Pro GlyMet Met Val Asp Ser Thr Phe Tyr Asn Lys Ile Lys Ser Gly 660 665 670 TyrArg Met Ala Lys Pro Asp His Ala Thr Ser Glu Val Tyr Glu Ile 675 680 685Met Val Lys Cys Trp Asn Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr 690 695700 His Leu Ser Glu Ile Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys 705710 715 720 Ser Tyr Glu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His ProAla 725 730 735 Val Ala Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile GlyVal Thr 740 745 750 Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu GlyGly Leu Asp 755 760 765 Glu Gln Arg Leu Ser Ala Asp Ser Gly Tyr Ile IlePro Leu Pro Asp 770 775 780 Ile Asp Pro Val Pro Glu Glu Glu Asp Leu GlyLys Arg Asn Arg His 785 790 795 800 Ser Ser Gln Thr Ser Glu Glu Ser AlaIle Glu Thr Gly Ser Ser Ser 805 810 815 Ser Thr Phe Ile Lys Arg Glu AspGlu Thr Ile Glu Asp Ile Asp Met 820 825 830 Met Asp Asp Ile Gly Ile AspSer Ser Asp Leu Val Glu Asp Ser Phe 835 840 845 Leu 2 834 PRT homosapiens 2 Met Ser Ala Gly Glu Val Glu Arg Leu Val Ser Glu Leu Ser GlyGly 1 5 10 15 Thr Gly Gly Asp Glu Glu Glu Glu Trp Leu Tyr Gly Asp GluAsn Glu 20 25 30 Val Glu Arg Pro Glu Glu Glu Asn Ala Ser Ala Asn Pro ProSer Gly 35 40 45 Ile Glu Asp Glu Thr Ala Glu Asn Gly Val Pro Lys Pro LysVal Thr 50 55 60 Glu Thr Glu Asp Asp Ser Asp Ser Asp Ser Asp Asp Asp GluAsp Asp 65 70 75 80 Val His Val Thr Ile Gly Asp Ile Lys Thr Gly Ala ProGln Tyr Gly 85 90 95 Ser Tyr Gly Thr Ala Pro Val Asn Leu Asn Ile Lys ThrGly Gly Arg 100 105 110 Val Tyr Gly Thr Thr Gly Thr Lys Val Lys Gly ValAsp Leu Asp Ala 115 120 125 Pro Gly Ser Ile Asn Gly Val Pro Leu Leu GluVal Asp Leu Asp Ser 130 135 140 Phe Glu Asp Lys Pro Trp Arg Lys Pro GlyAla Asp Leu Ser Asp Tyr 145 150 155 160 Phe Asn Tyr Gly Phe Asn Glu AspThr Trp Lys Ala Tyr Cys Glu Lys 165 170 175 Gln Lys Arg Ile Arg Met GlyLeu Glu Val Ile Pro Val Thr Ser Thr 180 185 190 Thr Asn Lys Ile Thr AlaGlu Asp Cys Thr Met Glu Val Thr Pro Gly 195 200 205 Ala Glu Ile Gln AspGly Arg Phe Asn Leu Phe Lys Val Gln Gln Gly 210 215 220 Arg Thr Gly AsnSer Glu Lys Glu Thr Ala Leu Pro Ser Thr Lys Ala 225 230 235 240 Glu PheThr Ser Pro Pro Ser Leu Phe Lys Thr Gly Leu Pro Pro Ser 245 250 255 ArgAsn Ser Thr Ser Ser Gln Ser Gln Thr Ser Thr Ala Ser Arg Lys 260 265 270Ala Asn Ser Ser Val Gly Lys Trp Gln Asp Arg Tyr Gly Arg Ala Glu 275 280285 Ser Pro Asp Leu Arg Arg Leu Pro Gly Ala Ile Asp Val Ile Gly Gln 290295 300 Thr Ile Thr Ile Ser Arg Val Glu Gly Arg Arg Arg Ala Asn Glu Asn305 310 315 320 Ser Asn Ile Gln Leu Pro Tyr Asp Ser Arg Trp Glu Phe ProArg Asp 325 330 335 Gly Leu Val Leu Gly Arg Val Leu Gly Ser Gly Ala PheGly Lys Val 340 345 350 Val Glu Gly Thr Ala Tyr Gly Leu Ser Arg Ser GlnPro Val Met Lys 355 360 365 Val Ala Val Lys Met Leu Lys Pro Thr Ala ArgSer Ser Glu Lys Gln 370 375 380 Ala Leu Met Ser Glu Leu Lys Ile Met ThrHis Leu Gly Pro His Leu 385 390 395 400 Asn Ile Val Asn Leu Leu Gly AlaCys Thr Lys Ser Gly Pro Ile Tyr 405 410 415 Ile Ile Thr Glu Tyr Cys PheTyr Gly Asp Leu Val Asn Tyr Leu His 420 425 430 Lys Asn Arg Asp Ser PheLeu Ser His His Pro Glu Lys Pro Lys Lys 435 440 445 Glu Leu Asp Ile PheGly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser 450 455 460 Tyr Val Ile LeuSer Phe Glu Asn Asn Gly Asp Tyr Met Asp Met Lys 465 470 475 480 Gln AlaAsp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys Glu Val 485 490 495 SerLys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp Arg Pro Ala Ser 500 505 510Tyr Lys Lys Lys Ser Met Leu Asp Ser Glu Val Lys Asn Leu Leu Ser 515 520525 Asp Asp Asn Ser Glu Gly Leu Thr Leu Leu Asp Leu Leu Ser Phe Thr 530535 540 Tyr Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Lys Asn Cys Val545 550 555 560 His Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Ala Gln GlyLys Ile 565 570 575 Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile MetHis Asp Ser 580 585 590 Asn Tyr Val Ser Lys Gly Ser Thr Phe Leu Pro ValLys Trp Met Ala 595 600 605 Pro Glu Ser Ile Phe Asp Asn Leu Tyr Thr ThrLeu Ser Asp Val Trp 610 615 620 Ser Tyr Gly Ile Leu Leu Trp Glu Ile PheSer Leu Gly Gly Thr Pro 625 630 635 640 Tyr Pro Gly Met Met Val Asp SerThr Phe Tyr Asn Lys Ile Lys Ser 645 650 655 Gly Tyr Arg Met Ala Lys ProAsp His Ala Thr Ser Glu Val Tyr Glu 660 665 670 Ile Met Val Lys Cys TrpAsn Ser Glu Pro Glu Lys Arg Pro Ser Phe 675 680 685 Tyr His Leu Ser GluIle Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys 690 695 700 Lys Ser Tyr GluLys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro 705 710 715 720 Ala ValAla Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly Val 725 730 735 ThrTyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu Gly Gly Leu 740 745 750Asp Glu Gln Arg Leu Ser Ala Asp Ser Gly Tyr Ile Ile Pro Leu Pro 755 760765 Asp Ile Asp Pro Val Pro Glu Glu Glu Asp Leu Gly Lys Arg Asn Arg 770775 780 His Ser Ser Gln Thr Ser Glu Glu Ser Ala Ile Glu Thr Gly Ser Ser785 790 795 800 Ser Ser Thr Phe Ile Lys Arg Glu Asp Glu Thr Ile Glu AspIle Asp 805 810 815 Met Met Asp Asp Ile Gly Ile Asp Ser Ser Asp Leu ValGlu Asp Ser 820 825 830 Phe Leu 3 826 PRT homo sapiens 3 Met Ser Ala GlyGlu Val Glu Arg Leu Val Ser Glu Leu Ser Gly Gly 1 5 10 15 Thr Gly GlyAsp Glu Glu Glu Glu Trp Leu Tyr Gly Gly Pro Trp Asp 20 25 30 Val His ValHis Ser Asp Leu Ala Lys Asp Leu Asp Glu Asn Glu Val 35 40 45 Glu Arg ProGlu Glu Glu Asn Ala Ser Ala Asn Pro Pro Ser Gly Ile 50 55 60 Glu Asp GluThr Ala Glu Asn Gly Val Pro Lys Pro Lys Val Thr Glu 65 70 75 80 Thr GluAsp Asp Ser Asp Ser Asp Ser Asp Asp Asp Glu Asp Asp Val 85 90 95 His ValThr Ile Gly Asp Ile Lys Thr Gly Ala Pro Gln Tyr Gly Ser 100 105 110 TyrGly Thr Ala Pro Val Asn Leu Asn Ile Lys Thr Gly Gly Arg Val 115 120 125Tyr Gly Thr Thr Gly Thr Lys Val Lys Gly Val Asp Leu Asp Ala Pro 130 135140 Gly Ser Ile Asn Gly Val Pro Leu Leu Glu Val Asp Leu Asp Ser Phe 145150 155 160 Glu Asp Lys Pro Trp Arg Lys Pro Gly Ala Asp Leu Ser Asp TyrPhe 165 170 175 Asn Tyr Gly Phe Asn Glu Asp Thr Trp Lys Ala Tyr Cys GluLys Gln 180 185 190 Lys Arg Ile Arg Met Gly Leu Glu Val Ile Pro Val ThrSer Thr Thr 195 200 205 Asn Lys Ile Thr Val Gln Gln Gly Arg Thr Gly AsnSer Glu Lys Glu 210 215 220 Thr Ala Leu Pro Ser Thr Lys Ala Glu Phe ThrSer Pro Pro Ser Leu 225 230 235 240 Phe Lys Thr Gly Leu Pro Pro Ser ArgAsn Ser Thr Ser Ser Gln Ser 245 250 255 Gln Thr Ser Thr Ala Ser Arg LysAla Asn Ser Ser Val Gly Lys Trp 260 265 270 Gln Asp Arg Tyr Gly Arg AlaGlu Ser Pro Asp Leu Arg Arg Leu Pro 275 280 285 Gly Ala Ile Asp Val IleGly Gln Thr Ile Thr Ile Ser Arg Val Glu 290 295 300 Gly Arg Arg Arg AlaAsn Glu Asn Ser Asn Ile Gln Leu Pro Tyr Asp 305 310 315 320 Ser Arg TrpGlu Phe Pro Arg Asp Gly Leu Val Leu Gly Arg Val Leu 325 330 335 Gly SerGly Ala Phe Gly Lys Val Val Glu Gly Thr Ala Tyr Gly Leu 340 345 350 SerArg Ser Gln Pro Val Met Lys Val Ala Val Lys Met Leu Lys Pro 355 360 365Thr Ala Arg Ser Ser Glu Lys Gln Ala Leu Met Ser Glu Leu Lys Ile 370 375380 Met Thr His Leu Gly Pro His Leu Asn Ile Val Asn Leu Leu Gly Ala 385390 395 400 Cys Thr Lys Ser Gly Pro Ile Tyr Ile Ile Thr Glu Tyr Cys PheTyr 405 410 415 Gly Asp Leu Val Asn Tyr Leu His Lys Asn Arg Asp Ser PheLeu Ser 420 425 430 His His Pro Glu Lys Pro Lys Lys Glu Leu Asp Ile PheGly Leu Asn 435 440 445 Pro Ala Asp Glu Ser Thr Arg Ser Tyr Val Ile LeuSer Phe Glu Asn 450 455 460 Asn Gly Asp Tyr Met Asp Met Lys Gln Ala AspThr Thr Gln Tyr Val 465 470 475 480 Pro Met Leu Glu Arg Lys Glu Val SerLys Tyr Ser Asp Ile Gln Arg 485 490 495 Ser Leu Tyr Asp Arg Pro Ala SerTyr Lys Lys Lys Ser Met Leu Asp 500 505 510 Ser Glu Val Lys Asn Leu LeuSer Asp Asp Asn Ser Glu Gly Leu Thr 515 520 525 Leu Leu Asp Leu Leu SerPhe Thr Tyr Gln Val Ala Arg Gly Met Glu 530 535 540 Phe Leu Ala Ser LysAsn Cys Val His Arg Asp Leu Ala Ala Arg Asn 545 550 555 560 Val Leu LeuAla Gln Gly Lys Ile Val Lys Ile Cys Asp Phe Gly Leu 565 570 575 Ala ArgAsp Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly Ser Thr 580 585 590 PheLeu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Asn Leu 595 600 605Tyr Thr Thr Leu Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu 610 615620 Ile Phe Ser Leu Gly Gly Thr Pro Tyr Pro Gly Met Met Val Asp Ser 625630 635 640 Thr Phe Tyr Asn Lys Ile Lys Ser Gly Tyr Arg Met Ala Lys ProAsp 645 650 655 His Ala Thr Ser Glu Val Tyr Glu Ile Met Val Lys Cys TrpAsn Ser 660 665 670 Glu Pro Glu Lys Arg Pro Ser Phe Tyr His Leu Ser GluIle Val Glu 675 680 685 Asn Leu Leu Pro Gly Gln Tyr Lys Lys Ser Tyr GluLys Ile His Leu 690 695 700 Asp Phe Leu Lys Ser Asp His Pro Ala Val AlaArg Met Arg Val Asp 705 710 715 720 Ser Asp Asn Ala Tyr Ile Gly Val ThrTyr Lys Asn Glu Glu Asp Lys 725 730 735 Leu Lys Asp Trp Glu Gly Gly LeuAsp Glu Gln Arg Leu Ser Ala Asp 740 745 750 Ser Gly Tyr Ile Ile Pro LeuPro Asp Ile Asp Pro Val Pro Glu Glu 755 760 765 Glu Asp Leu Gly Lys ArgAsn Arg His Ser Ser Gln Thr Ser Glu Glu 770 775 780 Ser Ala Ile Glu ThrGly Ser Ser Ser Ser Thr Phe Ile Lys Arg Glu 785 790 795 800 Asp Glu ThrIle Glu Asp Ile Asp Met Met Asp Asp Ile Gly Ile Asp 805 810 815 Ser SerAsp Leu Val Glu Asp Ser Phe Leu 820 825 4 811 PRT homo sapiens 4 Met SerAla Gly Glu Val Glu Arg Leu Val Ser Glu Leu Ser Gly Gly 1 5 10 15 ThrGly Gly Asp Glu Glu Glu Glu Trp Leu Tyr Gly Asp Glu Asn Glu 20 25 30 ValGlu Arg Pro Glu Glu Glu Asn Ala Ser Ala Asn Pro Pro Ser Gly 35 40 45 IleGlu Asp Glu Thr Ala Glu Asn Gly Val Pro Lys Pro Lys Val Thr 50 55 60 GluThr Glu Asp Asp Ser Asp Ser Asp Ser Asp Asp Asp Glu Asp Asp 65 70 75 80Val His Val Thr Ile Gly Asp Ile Lys Thr Gly Ala Pro Gln Tyr Gly 85 90 95Ser Tyr Gly Thr Ala Pro Val Asn Leu Asn Ile Lys Thr Gly Gly Arg 100 105110 Val Tyr Gly Thr Thr Gly Thr Lys Val Lys Gly Val Asp Leu Asp Ala 115120 125 Pro Gly Ser Ile Asn Gly Val Pro Leu Leu Glu Val Asp Leu Asp Ser130 135 140 Phe Glu Asp Lys Pro Trp Arg Lys Pro Gly Ala Asp Leu Ser AspTyr 145 150 155 160 Phe Asn Tyr Gly Phe Asn Glu Asp Thr Trp Lys Ala TyrCys Glu Lys 165 170 175 Gln Lys Arg Ile Arg Met Gly Leu Glu Val Ile ProVal Thr Ser Thr 180 185 190 Thr Asn Lys Ile Thr Val Gln Gln Gly Arg ThrGly Asn Ser Glu Lys 195 200 205 Glu Thr Ala Leu Pro Ser Thr Lys Ala GluPhe Thr Ser Pro Pro Ser 210 215 220 Leu Phe Lys Thr Gly Leu Pro Pro SerArg Asn Ser Thr Ser Ser Gln 225 230 235 240 Ser Gln Thr Ser Thr Ala SerArg Lys Ala Asn Ser Ser Val Gly Lys 245 250 255 Trp Gln Asp Arg Tyr GlyArg Ala Glu Ser Pro Asp Leu Arg Arg Leu 260 265 270 Pro Gly Ala Ile AspVal Ile Gly Gln Thr Ile Thr Ile Ser Arg Val 275 280 285 Glu Gly Arg ArgArg Ala Asn Glu Asn Ser Asn Ile Gln Leu Pro Tyr 290 295 300 Asp Ser ArgTrp Glu Phe Pro Arg Asp Gly Leu Val Leu Gly Arg Val 305 310 315 320 LeuGly Ser Gly Ala Phe Gly Lys Val Val Glu Gly Thr Ala Tyr Gly 325 330 335Leu Ser Arg Ser Gln Pro Val Met Lys Val Ala Val Lys Met Leu Lys 340 345350 Pro Thr Ala Arg Ser Ser Glu Lys Gln Ala Leu Met Ser Glu Leu Lys 355360 365 Ile Met Thr His Leu Gly Pro His Leu Asn Ile Val Asn Leu Leu Gly370 375 380 Ala Cys Thr Lys Ser Gly Pro Ile Tyr Ile Ile Thr Glu Tyr CysPhe 385 390 395 400 Tyr Gly Asp Leu Val Asn Tyr Leu His Lys Asn Arg AspSer Phe Leu 405 410 415 Ser His His Pro Glu Lys Pro Lys Lys Glu Leu AspIle Phe Gly Leu 420 425 430 Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr ValIle Leu Ser Phe Glu 435 440 445 Asn Asn Gly Asp Tyr Met Asp Met Lys GlnAla Asp Thr Thr Gln Tyr 450 455 460 Val Pro Met Leu Glu Arg Lys Glu ValSer Lys Tyr Ser Asp Ile Gln 465 470 475 480 Arg Ser Leu Tyr Asp Arg ProAla Ser Tyr Lys Lys Lys Ser Met Leu 485 490 495 Asp Ser Glu Val Lys AsnLeu Leu Ser Asp Asp Asn Ser Glu Gly Leu 500 505 510 Thr Leu Leu Asp LeuLeu Ser Phe Thr Tyr Gln Val Ala Arg Gly Met 515 520 525 Glu Phe Leu AlaSer Lys Asn Cys Val His Arg Asp Leu Ala Ala Arg 530 535 540 Asn Val LeuLeu Ala Gln Gly Lys Ile Val Lys Ile Cys Asp Phe Gly 545 550 555 560 LeuAla Arg Asp Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly Ser 565 570 575Thr Phe Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Asn 580 585590 Leu Tyr Thr Thr Leu Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp 595600 605 Glu Ile Phe Ser Leu Gly Gly Thr Pro Tyr Pro Gly Met Met Val Asp610 615 620 Ser Thr Phe Tyr Asn Lys Ile Lys Ser Gly Tyr Arg Met Ala LysPro 625 630 635 640 Asp His Ala Thr Ser Glu Val Tyr Glu Ile Met Val LysCys Trp Asn 645 650 655 Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr His LeuSer Glu Ile Val 660 665 670 Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys SerTyr Glu Lys Ile His 675 680 685 Leu Asp Phe Leu Lys Ser Asp His Pro AlaVal Ala Arg Met Arg Val 690 695 700 Asp Ser Asp Asn Ala Tyr Ile Gly ValThr Tyr Lys Asn Glu Glu Asp 705 710 715 720 Lys Leu Lys Asp Trp Glu GlyGly Leu Asp Glu Gln Arg Leu Ser Ala 725 730 735 Asp Ser Gly Tyr Ile IlePro Leu Pro Asp Ile Asp Pro Val Pro Glu 740 745 750 Glu Glu Asp Leu GlyLys Arg Asn Arg His Ser Ser Gln Thr Ser Glu 755 760 765 Glu Ser Ala IleGlu Thr Gly Ser Ser Ser Ser Thr Phe Ile Lys Arg 770 775 780 Glu Asp GluThr Ile Glu Asp Ile Asp Met Met Asp Asp Ile Gly Ile 785 790 795 800 AspSer Ser Asp Leu Val Glu Asp Ser Phe Leu 805 810 5 2541 DNA homo sapiens5 atgtcggccg gcgaggtcga gcgcctagtg tcggagctga gcggcgggac cggaggggat 60gaggaggaag agtggctcta tggcggccca tgggacgtgc atgtgcacag tgatttggca 120aaggacctag atgaaaatga agttgaaagg ccagaagaag aaaatgccag tgctaatcct 180ccatctggaa ttgaagatga aactgctgaa aatggtgtac caaaaccgaa agtgactgag 240accgaagatg atagtgatag tgacagcgat gatgatgaag atgatgttca tgtcactata 300ggagacatta aaacgggagc accacagtat gggagttatg gtacagcacc tgtaaatctt 360aacatcaaga cagggggaag agtttatgga actacaggga caaaagtcaa aggagtagac 420cttgatgcac ctggaagcat taatggagtt ccactcttag aggtagattt ggattctttt 480gaagataaac catggcgtaa acctggtgct gatctttctg attattttaa ttatgggttt 540aatgaagata cctggaaagc ttactgtgaa aaacaaaaga ggatacgaat gggacttgaa 600gttataccag taacctctac tacaaataaa attacggccg aagactgtac tatggaagtt 660acaccaggtg cagagatcca agatggcaga ttcaatcttt ttaaggtaca gcagggaaga 720actggaaact cagagaaaga aactgccctt ccatctacaa aagctgagtt tacttctcct 780ccttctttgt tcaagactgg gcttccaccg agcagaaaca gcacttcttc tcagtctcag 840acaagtactg cctccagaaa agccaattca agcgttggga agtggcagga tcgatatggg 900agggccgaat cacctgatct aaggagatta cctggggcaa ttgatgttat cggtcagact 960ataactatca gccgagtaga aggcaggcga cgggcaaatg agaacagcaa catacaggac 1020tcaagatggg agtttccaag agatggacta gtgcttggtc gggtcttggg gtctggagcg 1080tttgggaagg tggttgaagg aacagcctat ggattaagcc ggtcccaacc tgtcatgaaa 1140gttgcagtga agatgctaaa acccacggcc agatccagtg aaaaacaagc tctcatgtct 1200gaactgaaga taatgactca cctggggcca catttgaaca ttgtaaactt gctgggagcc 1260tgcaccaagt caggccccat ttacatcatc acagagtatt gcttctatgg agatttggtc 1320aactatttgc ataagaatag ggatagcttc ctgagccacc acccagagaa gccaaagaaa 1380gagctggata tctttggatt gaaccctgct gatgaaagca cacggagcta tgttatttta 1440tcttttgaaa acaatggtga ctacatggac atgaagcagg ctgatactac acagtatgtc 1500cccatgctag aaaggaaaga ggtttctaaa tattccgaca tccagagatc actctatgat 1560cgtccagcct catataagaa gaaatctatg ttagactcag aagtcaaaaa cctcctttca 1620gatgataact cagaaggcct tactttattg gatttgttga gcttcaccta tcaagttgcc 1680cgaggaatgg agtttttggc ttcaaaaaat tgtgtccacc gtgatctggc tgctcgcaac 1740gttctcctgg cacaaggaaa aattgtgaag atctgtgact ttggcctggc cagagacatc 1800atgcatgatt cgaactatgt gtcgaaaggc agtacctttc tgcccgtgaa gtggatggct 1860cctgagagca tctttgacaa cctctacacc acactgagtg atgtctggtc ttatggcatt 1920ctgctctggg agatcttttc ccttggtggc accccttacc ccggcatgat ggtggattct 1980actttctaca ataagatcaa gagtgggtac cggatggcca agcctgacca cgctaccagt 2040gaagtctacg agatcatggt gaaatgctgg aacagtgagc cggagaagag accctccttt 2100taccacctga gtgagattgt ggagaatctg ctgcctggac aatataaaaa gagttatgaa 2160aaaattcacc tggacttcct gaagagtgac catcctgctg tggcacgcat gcgtgtggac 2220tcagacaatg catacattgg tgtcacctac aaaaacgagg aagacaagct gaaggactgg 2280gagggtggtc tggatgagca gagactgagc gctgacagtg gctacatcat tcctctgcct 2340gacattgacc ctgtccctga ggaggaggac ctgggcaaga ggaacagaca cagctcgcag 2400acctctgaag agagtgccat tgagacgggt tccagcagtt ccaccttcat caagagagag 2460gacgagacca ttgaagacat cgacatgatg gacgacatcg gcatagactc ttcagacctg 2520gtggaagaca gcttcctgta a 2541 6 2496 DNA homo sapiens 6 atgtcggccggcgaggtcga gcgcctagtg tcggagctga gcggcgggac cggaggggat 60 gaggaggaagagtggctcta tggcgatgaa aatgaagttg aaaggccaga agaagaaaat 120 gccagtgctaatcctccatc tggaattgaa gatgaaactg ctgaaaatgg tgtaccaaaa 180 ccgaaagtgactgagaccga agatgatagt gatagtgaca gcgatgatga tgaagatgat 240 gttcatgtcactataggaga cattaaaacg ggagcaccac agtatgggag ttatggtaca 300 gcacctgtaaatcttaacat caagacaggg ggaagagttt atggaactac agggacaaaa 360 gtcaaaggagtagaccttga tgcacctgga agcattaatg gagttccact cttagaggta 420 gatttggattcttttgaaga taaaccatgg cgtaaacctg gtgctgatct ttctgattat 480 tttaattatgggtttaatga agatacctgg aaagcttact gtgaaaaaca aaagaggata 540 cgaatgggacttgaagttat accagtaacc tctactacaa ataaaattac ggccgaagac 600 tgtactatggaagttacacc aggtgcagag atccaagatg gcagattcaa tctttttaag 660 gtacagcagggaagaactgg aaactcagag aaagaaactg cccttccatc tacaaaagct 720 gagtttacttctcctccttc tttgttcaag actgggcttc caccgagcag aaacagcact 780 tcttctcagtctcagacaag tactgcctcc agaaaagcca attcaagcgt tgggaagtgg 840 caggatcgatatgggagggc cgaatcacct gatctaagga gattacctgg ggcaattgat 900 gttatcggtcagactataac tatcagccga gtagaaggca ggcgacgggc aaatgagaac 960 agcaacatacaggactcaag atgggagttt ccaagagatg gactagtgct tggtcgggtc 1020 ttggggtctggagcgtttgg gaaggtggtt gaaggaacag cctatggatt aagccggtcc 1080 caacctgtcatgaaagttgc agtgaagatg ctaaaaccca cggccagatc cagtgaaaaa 1140 caagctctcatgtctgaact gaagataatg actcacctgg ggccacattt gaacattgta 1200 aacttgctgggagcctgcac caagtcaggc cccatttaca tcatcacaga gtattgcttc 1260 tatggagatttggtcaacta tttgcataag aatagggata gcttcctgag ccaccaccca 1320 gagaagccaaagaaagagct ggatatcttt ggattgaacc ctgctgatga aagcacacgg 1380 agctatgttattttatcttt tgaaaacaat ggtgactaca tggacatgaa gcaggctgat 1440 actacacagtatgtccccat gctagaaagg aaagaggttt ctaaatattc cgacatccag 1500 agatcactctatgatcgtcc agcctcatat aagaagaaat ctatgttaga ctcagaagtc 1560 aaaaacctcctttcagatga taactcagaa ggccttactt tattggattt gttgagcttc 1620 acctatcaagttgcccgagg aatggagttt ttggcttcaa aaaattgtgt ccaccgtgat 1680 ctggctgctcgcaacgttct cctggcacaa ggaaaaattg tgaagatctg tgactttggc 1740 ctggccagagacatcatgca tgattcgaac tatgtgtcga aaggcagtac ctttctgccc 1800 gtgaagtggatggctcctga gagcatcttt gacaacctct acaccacact gagtgatgtc 1860 tggtcttatggcattctgct ctgggagatc ttttcccttg gtggcacccc ttaccccggc 1920 atgatggtggattctacttt ctacaataag atcaagagtg ggtaccggat ggccaagcct 1980 gaccacgctaccagtgaagt ctacgagatc atggtgaaat gctggaacag tgagccggag 2040 aagagaccctccttttacca cctgagtgag attgtggaga atctgctgcc tggacaatat 2100 aaaaagagttatgaaaaaat tcacctggac ttcctgaaga gtgaccatcc tgctgtggca 2160 cgcatgcgtgtggactcaga caatgcatac attggtgtca cctacaaaaa cgaggaagac 2220 aagctgaaggactgggaggg tggtctggat gagcagagac tgagcgctga cagtggctac 2280 atcattcctctgcctgacat tgaccctgtc cctgaggagg aggacctggg caagaggaac 2340 agacacagctcgcagacctc tgaagagagt gccattgaga cgggttccag cagttccacc 2400 ttcatcaagagagaggacga gaccattgaa gacatcgaca tgatggacga catcggcata 2460 gactcttcagacctggtgga agacagcttc ctgtaa 2496 7 2472 DNA homo sapiens 7 atgtcggccggcgaggtcga gcgcctagtg tcggagctga gcggcgggac cggaggggat 60 gaggaggaagagtggctcta tggcggccca tgggacgtgc atgtgcacag tgatttggca 120 aaggacctagatgaaaatga agttgaaagg ccagaagaag aaaatgccag tgctaatcct 180 ccatctggaattgaagatga aactgctgaa aatggtgtac caaaaccgaa agtgactgag 240 accgaagatgatagtgatag tgacagcgat gatgatgaag atgatgttca tgtcactata 300 ggagacattaaaacgggagc accacagtat gggagttatg gtacagcacc tgtaaatctt 360 aacatcaagacagggggaag agtttatgga actacaggga caaaagtcaa aggagtagac 420 cttgatgcacctggaagcat taatggagtt ccactcttag aggtagattt ggattctttt 480 gaagataaaccatggcgtaa acctggtgct gatctttctg attattttaa ttatgggttt 540 aatgaagatacctggaaagc ttactgtgaa aaacaaaaga ggatacgaat gggacttgaa 600 gttataccagtaacctctac tacaaataaa attacggtac agcagggaag aactggaaac 660 tcagagaaagaaactgccct tccatctaca aaagctgagt ttacttctcc tccttctttg 720 ttcaagactgggcttccacc gagcagaaac agcacttctt ctcagtctca gacaagtact 780 gcctccagaaaagccaattc aagcgttggg aagtggcagg atcgatatgg gagggccgaa 840 tcacctgatctaaggagatt acctggggca attgatgtta tcggtcagac tataactatc 900 agccgagtagaaggcaggcg acgggcaaat gagaacagca acatacagga ctcaagatgg 960 gagtttccaagagatggact agtgcttggt cgggtcttgg ggtctggagc gtttgggaag 1020 gtggttgaaggaacagccta tggattaagc cggtcccaac ctgtcatgaa agttgcagtg 1080 aagatgctaaaacccacggc cagatccagt gaaaaacaag ctctcatgtc tgaactgaag 1140 ataatgactcacctggggcc acatttgaac attgtaaact tgctgggagc ctgcaccaag 1200 tcaggccccatttacatcat cacagagtat tgcttctatg gagatttggt caactatttg 1260 cataagaatagggatagctt cctgagccac cacccagaga agccaaagaa agagctggat 1320 atctttggattgaaccctgc tgatgaaagc acacggagct atgttatttt atcttttgaa 1380 aacaatggtgactacatgga catgaagcag gctgatacta cacagtatgt ccccatgcta 1440 gaaaggaaagaggtttctaa atattccgac atccagagat cactctatga tcgtccagcc 1500 tcatataagaagaaatctat gttagactca gaagtcaaaa acctcctttc agatgataac 1560 tcagaaggccttactttatt ggatttgttg agcttcacct atcaagttgc ccgaggaatg 1620 gagtttttggcttcaaaaaa ttgtgtccac cgtgatctgg ctgctcgcaa cgttctcctg 1680 gcacaaggaaaaattgtgaa gatctgtgac tttggcctgg ccagagacat catgcatgat 1740 tcgaactatgtgtcgaaagg cagtaccttt ctgcccgtga agtggatggc tcctgagagc 1800 atctttgacaacctctacac cacactgagt gatgtctggt cttatggcat tctgctctgg 1860 gagatcttttcccttggtgg caccccttac cccggcatga tggtggattc tactttctac 1920 aataagatcaagagtgggta ccggatggcc aagcctgacc acgctaccag tgaagtctac 1980 gagatcatggtgaaatgctg gaacagtgag ccggagaaga gaccctcctt ttaccacctg 2040 agtgagattgtggagaatct gctgcctgga caatataaaa agagttatga aaaaattcac 2100 ctggacttcctgaagagtga ccatcctgct gtggcacgca tgcgtgtgga ctcagacaat 2160 gcatacattggtgtcaccta caaaaacgag gaagacaagc tgaaggactg ggagggtggt 2220 ctggatgagcagagactgag cgctgacagt ggctacatca ttcctctgcc tgacattgac 2280 cctgtccctgaggaggagga cctgggcaag aggaacagac acagctcgca gacctctgaa 2340 gagagtgccattgagacggg ttccagcagt tccaccttca tcaagagaga ggacgagacc 2400 attgaagacatcgacatgat ggacgacatc ggcatagact cttcagacct ggtggaagac 2460 agcttcctgtaa 2472 8 2427 DNA homo sapiens 8 atgtcggccg gcgaggtcga gcgcctagtgtcggagctga gcggcgggac cggaggggat 60 gaggaggaag agtggctcta tggcgatgaaaatgaagttg aaaggccaga agaagaaaat 120 gccagtgcta atcctccatc tggaattgaagatgaaactg ctgaaaatgg tgtaccaaaa 180 ccgaaagtga ctgagaccga agatgatagtgatagtgaca gcgatgatga tgaagatgat 240 gttcatgtca ctataggaga cattaaaacgggagcaccac agtatgggag ttatggtaca 300 gcacctgtaa atcttaacat caagacagggggaagagttt atggaactac agggacaaaa 360 gtcaaaggag tagaccttga tgcacctggaagcattaatg gagttccact cttagaggta 420 gatttggatt cttttgaaga taaaccatggcgtaaacctg gtgctgatct ttctgattat 480 tttaattatg ggtttaatga agatacctggaaagcttact gtgaaaaaca aaagaggata 540 cgaatgggac ttgaagttat accagtaacctctactacaa ataaaattac ggtacagcag 600 ggaagaactg gaaactcaga gaaagaaactgcccttccat ctacaaaagc tgagtttact 660 tctcctcctt ctttgttcaa gactgggcttccaccgagca gaaacagcac ttcttctcag 720 tctcagacaa gtactgcctc cagaaaagccaattcaagcg ttgggaagtg gcaggatcga 780 tatgggaggg ccgaatcacc tgatctaaggagattacctg gggcaattga tgttatcggt 840 cagactataa ctatcagccg agtagaaggcaggcgacggg caaatgagaa cagcaacata 900 caggactcaa gatgggagtt tccaagagatggactagtgc ttggtcgggt cttggggtct 960 ggagcgtttg ggaaggtggt tgaaggaacagcctatggat taagccggtc ccaacctgtc 1020 atgaaagttg cagtgaagat gctaaaacccacggccagat ccagtgaaaa acaagctctc 1080 atgtctgaac tgaagataat gactcacctggggccacatt tgaacattgt aaacttgctg 1140 ggagcctgca ccaagtcagg ccccatttacatcatcacag agtattgctt ctatggagat 1200 ttggtcaact atttgcataa gaatagggatagcttcctga gccaccaccc agagaagcca 1260 aagaaagagc tggatatctt tggattgaaccctgctgatg aaagcacacg gagctatgtt 1320 attttatctt ttgaaaacaa tggtgactacatggacatga agcaggctga tactacacag 1380 tatgtcccca tgctagaaag gaaagaggtttctaaatatt ccgacatcca gagatcactc 1440 tatgatcgtc cagcctcata taagaagaaatctatgttag actcagaagt caaaaacctc 1500 ctttcagatg ataactcaga aggccttactttattggatt tgttgagctt cacctatcaa 1560 gttgcccgag gaatggagtt tttggcttcaaaaaattgtg tccaccgtga tctggctgct 1620 cgcaacgttc tcctggcaca aggaaaaattgtgaagatct gtgactttgg cctggccaga 1680 gacatcatgc atgattcgaa ctatgtgtcgaaaggcagta cctttctgcc cgtgaagtgg 1740 atggctcctg agagcatctt tgacaacctctacaccacac tgagtgatgt ctggtcttat 1800 ggcattctgc tctgggagat cttttcccttggtggcaccc cttaccccgg catgatggtg 1860 gattctactt tctacaataa gatcaagagtgggtaccgga tggccaagcc tgaccacgct 1920 accagtgaag tctacgagat catggtgaaatgctggaaca gtgagccgga gaagagaccc 1980 tccttttacc acctgagtga gattgtggagaatctgctgc ctggacaata taaaaagagt 2040 tatgaaaaaa ttcacctgga cttcctgaagagtgaccatc ctgctgtggc acgcatgcgt 2100 gtggactcag acaatgcata cattggtgtcacctacaaaa acgaggaaga caagctgaag 2160 gactgggagg gtggtctgga tgagcagagactgagcgctg acagtggcta catcattcct 2220 ctgcctgaca ttgaccctgt ccctgaggaggaggacctgg gcaagaggaa cagacacagc 2280 tcgcagacct ctgaagagag tgccattgagacgggttcca gcagttccac cttcatcaag 2340 agagaggacg agaccattga agacatcgacatgatggacg acatcggcat agactcttca 2400 gacctggtgg aagacagctt cctgtaa 24279 1079 DNA homo sapiens 9 ggagattacc tggggcaatt gatgttatcg gtcagactataactatcagc cgagtagaag 60 gcaggcgacg ggcaaatgag aacagcaaca tacaggtttagtattttaaa ataaataatt 120 ttctttaact gaaggatgat atcaacatta taatttaatttattcaatag atagtcatag 180 tgctatgctg tgttttaggt gatacaaaga gatttaaaatgtgaagccag tcatttatta 240 taaaggcatt tccagttaga atttataatc tctgaatctttttttaatag ggttttcttt 300 cttttccttg gtagaaacat agtacaggtt gagtatctcttatctgaaat gcttgggacc 360 agaagtgtat cagattttga aatgtttgga ttttgaaatgttcatacgta tgtaatctta 420 gagatgagac ccaagtctaa acatgaaatt tatgtttcataaataagtta tttaatttgt 480 tataatgatt tgttaattta tgataatctt ttttcctttggcaaccctga gtaaactgtg 540 tagtgggcct gagttttgat tgtgatccgt cacatgtgaggttgggtgtg gaattttcca 600 cttgtgataa aatgttgatg ctcaaaatgt ttcggattttagagcatttc agatttcaga 660 tttttggatt aggaatactc aacttgtata taggtttactgaatgaaaaa taaaagacca 720 ccagtgattt taccacctaa agataaccac atctggtacatctcttgata ctaagcaaaa 780 ttgggatact attataatta tatttctgtg tatacttttttccccatcta aaattatcat 840 ttgtatgttt tcatattctt aaaatatgat taaaatatcttcatgatagc ttcattggat 900 aaatatacca tgatttattt aatgtgacat ttctggaggtggcgtagagc tgctttgttt 960 ttaggtgaaa aattgagggg agataaatta tcagtaagttggataattaa tgttagaact 1020 ttaataactg agacttccag ctattcattt tggccatactttttttcatt atttattcc 1079 10 770 DNA homo sapiens 10 tggtttgagagatggtactg cctatcccta aaatgaacca ggcagccctc acacttcccc 60 accagcagtgagagattcct ggctcagaca cagccacact accttgctgc ccctgtgcat 120 gtctgccaggaaacttttca ttgtgcctct ctctcttgtc acgtagccct gcgttctgaa 180 ctcacggtggctgctgcagt cctggtgctg ttggtgattg tgatcatctc acttattgtc 240 ctggttgtcatttggaaaca ggtagatatt ttctcataaa actaaagatc tttgaagcca 300 atgagaacaagcatagcaac ctagttcagt gcttggcaca gagaaggagc tcagcaatta 360 catgtggagtgaacgttgtt ggactctact gtgtccagtc actgtgctgc ttcagtgaag 420 ctctggtgcactgggacttt ggtaattcac cagttacctg tcctggtcat ttatagaaac 480 cgaggtatgaaattcgctgg agggtcattg aatcaatcag cccagatgga catgaatata 540 tttatgtggacccgatgcag ctgccttatg actcaagatg ggagtttcca agagatggac 600 tagtgcttggtaagttccat ggggtaacct cccaagactc ccttttccct tgcacacaac 660 tttacaatttataggccttg gcagaataga gatctgagct tgtgcttagt aagaactagg 720 caatggaaatttgctttcag aaatacattt ctgtcttgac agtaagttaa 770 11 12 DNA homo sapiens11 atacaggttt ag 12 12 18 DNA homo sapiens 12 gacccgatgc agctgcct 18 1320 DNA homo sapiens 13 tccgcattgc aataaagtgg 20 14 20 DNA homo sapiens14 gttgcgctcg gggcggccat 20 15 20 DNA homo sapiens 15 ttctgaacgggatccagagg 20 16 20 DNA homo sapiens 16 ggatgtcgga atatttagaa 20 17 20DNA homo sapiens 17 gcagaaaggt actgcctttc 20 18 19 DNA homo sapiens 18aattatgggt ttaatgaag 19 19 19 DNA homo sapiens 19 aactttcatg acaggttgg19 20 25 DNA homo sapiens 20 ttcttactaa gcacaagctc agatc 25 21 23 DNAhomo sapiens 21 aagcatctaa ttaggtgaaa ctg 23 22 20 DNA homo sapiens 22cagggaagaa ctggaaactc 20

What is claimed is:
 1. A fusion polypeptide comprising a C-terminaltyrosine kinase domain and an N-terminal domain wherein said fusionpolypeptide has a constitutively activated kinase activity, is presentin other than its naturally occurring environment and is furthercharacterized by at least one of the following features: (a) saidC-terminal domain is from a chromosome 4 encoded protein; (b) saidN-terminal domain is from a chromosome 4 encoded protein; and (c) saidfusion protein is not a product of a translocation event.
 2. The fusionpolypeptide according to claim 1, wherein said C-terminal domain is froma chromosome 4 encoded protein.
 3. The fusion polypeptide according toclaim 1, wherein said N-terminal domain is from a chromosome 4 encodedprotein.
 4. The fusion polypeptide according to claim 1, wherein saidfusion protein is not a product of a translocation event.
 5. The fusionpolypeptide according to claim 1, wherein said chromosome 4 tyrosinekinase domain comprises the C-terminal domain of a tyrosine kinasechosen from PDGFRα, c-Kit and VEGFR-2.
 6. The fusion polypeptideaccording to claim 1, wherein said tyrosine kinase is PDGFRα.
 7. Thefusion polypeptide according to claim 1, wherein said N-terminal domainis a NM_(—)030917 domain.
 8. The fusion polypeptide according to claim1, wherein said fusion polypeptide is a primate polypeptide.
 9. Thefusion polypeptide according to claim 8, wherein said primatepolypeptide is a human polypeptide.
 10. The fusion polypeptide accordingto claim 1, wherein said fusion polypeptide has an amino acid sequencethat is substantially the same as or identical to a sequence chosen fromSEQ ID NO:01, SEQ ID NO:02; SEQ ID NO:03 and SEQ ID NO:04.
 11. Thefusion polypeptide according to claim 1, wherein said fusion polypeptideis substantially pure.
 12. A polynucleotide present in other than itsnaturally occurring environment encoding a fusion polypeptide accordingto any one of claims 1 to
 11. 13. A polynucleotide according to claim 12having a sequence of residues that is substantially the same as oridentical to a sequence chosen from SEQ ID NO:05, SEQ ID NO:06; SEQ IDNO:07 and SEQ ID NO:08 and complements thereof.
 14. An expressioncassette comprising a polynucleotide present in other than its naturallyoccurring environment encoding a fusion polypeptide according toclaim
 1. 15. A cell comprising an expression cassette, wherein theexpression cassette comprises a polynucleotide present in other than itsnaturally occurring environment encoding a fusion polypeptide accordingto claim
 1. 16. A method of producing a fusion polypeptide according toclaim 1, said method comprising: (a) growing a cell comprising anexpression cassette, wherein the expression cassette comprises apolynucleotide present in other than its naturally occurring environmentencoding a fusion polypeptide according to claim 1, to express saidpolypeptide; and (b) harvesting said polypeptide.
 17. An antibody thatspecifically binds to a fusion polypeptide according to claim
 1. 18. Theantibody according to claim 17, wherein said antibody is a polyclonalantibody.
 19. The antibody according to claim 17, wherein said antibodyis a monoclonal antibody.
 20. A method of determining whether a hostsuffers from a disease condition, said method comprising: (a) assayingsaid host for the presence of a fusion polypeptide according to claim 1or a polynucleotide comprising a coding sequence therefore to obtain anassay result; and (b) employing said assay result to determine whethersaid host suffers from said disease condition.
 21. The method accordingto claim 20, wherein said disease condition is a hyperproliferativedisease condition.
 22. The method according to claim 20, wherein saidmethod further comprises obtaining a sample from said host.
 23. Themethod according to claim 20, wherein said assaying comprises employingan antibody that specifically binds to the fusion polypeptide.
 24. Themethod according to claim 22, wherein said assaying comprises screeningsaid sample for the presence of the polynucleotide.
 25. The methodaccording to claim 22, wherein said assaying comprises screening saidsample for a chromosomal deletion that produces the polynucleotide. 26.A method of identifying an agent that inhibits the activity of a fusionpolypeptide according to claim 1, said method comprising: contactingsaid fusion polypeptide with a test agent; and determining the effect,if any, of the test agent on the activity of said fusion polypeptide.27. The method according to claim 26, wherein said method is an in vivomethod.
 28. The method according to claim 26, wherein said method is anin vitro method.
 29. A method of identifying an agent that inhibitsexpression of a fusion polypeptide according to claim 1 from a codingsequence therefore, said method comprising: contacting said codingsequence with a test agent; and determining the effect, if any, of saidtest agent on the expression of said fusion polypeptide from said codingsequence.
 30. The method according to claim 29, wherein said method isan in vivo method.
 31. The method according to claim 29, wherein saidmethod is an in vitro method.
 32. An in vitro method for reducing theactivity of a fusion polypeptide according to claim 1, said methodcomprising contacting said fusion polypeptide with an agent that reducesthe tyrosine kinase activity of said fusion polypeptide.
 33. A method ofreducing the activity of a fusion polypeptide according to claim 1 in ahost, said method comprising: administering to said host an effectiveamount of an agent that reduces the tyrosine kinase activity of saidfusion polypeptide, with the proviso that when said agent is imatinibmesylate, said agent is administered to said host at a dosage that doesnot exceed about 300 mg/day.
 34. The method according to claim 33,wherein said agent is not imatinib mesylate.
 35. The method according toclaim 33, wherein said agent is selected from the group consisting ofPTK787, THRX-165724, MLN518, and SU11248.
 36. The method according toclaim 33, wherein said agent is imatinib mesylate.
 37. The methodaccording to claim 33, wherein said method is a method of treating adisease condition.
 38. The method according to claim 37, wherein saiddisease condition is a hyperproliferative disease condition.
 39. Amethod of treating a host suffering from a disease condition, saidmethod comprising: (a) diagnosing whether said host suffers from saiddisease condition using a method according to claim 20 to obtain adiagnostic result; and (b) treating said host employing a treatmentprotocol chosen based on said diagnostic result.
 40. The methodaccording to claim 39, wherein said treatment protocol comprisesadministering an effective amount of imatinib mesylate to said host. 41.The method according to claim 39, wherein said treatment protocolcomprises administering an effective amount of an agent selected fromthe group consisting of PTK787, THRX-165724, MLN518, and SU11248 to saidhost.
 42. A method of screening a sample for the presence of a tyrosinekinase fusion protein in said sample, said method comprising: (a)separating tyrosine-phosphorylated proteins from the remainingconstituents of said sample to produce a population of sample derivedtyrosine phosphorylated proteins; and (b) evaluating the constituentmembers of said population of sample derived tyrosine phosphorylatedproteins for the presence of domains from two or more differentproteins.
 43. The method according to claim 42, wherein said evaluatingstep (b) comprises sequencing at least two different domains of at leastone constituent member of said population.
 44. The method according toclaim 43, wherein said at least two different domains are the N andC-terminal domains.
 45. A method of characterizing a tyrosine kinasefusion protein identified in a screening assay according to claim 42,said method comprising: (a) obtaining a nucleic acid encoding saididentified tyrosine kinase fusion protein from said sample; and (b)determining the sequence of said encoding nucleic acid.
 46. The methodaccording to claim 45, wherein said method further comprises determiningthe amino acid sequence of the product encoded by said encoding nucleicacid.
 47. The method according to claim 45, wherein said method furthercomprises determining the sequence of genomic DNA that is transcribedinto said encoding nucleic acid.
 48. The method according to claim 42,wherein said sample is a cell.
 49. A non-human homolog of a fusionpolypeptide according to claim
 8. 50. The fusion polypeptide accordingto claim 49, wherein said fusion polypeptide is a mammalian fusionpolypeptide
 51. The fusion polypeptide according to claim 50, whereinsaid mammalian fusion polypeptide is a rodent fusion polypeptide.
 52. Aprocess for preparing a composition, the process comprising admixing apharmaceutically acceptable carrier and an agent that inhibits theactivity of a fusion polypeptide according to claim 1, wherein the agentis identified by a method comprising contacting said fusion polypeptidewith a test agent, and determining the effect, if any, of the test agenton the activity of said fusion polypeptide.
 53. A process for preparinga composition, the process comprising admixing a pharmaceuticallyacceptable carrier and an agent that inhibits the activity of a fusionpolypeptide according to claim 1, wherein the agent is identified by amethod comprising contacting a coding sequence for said fusionpolypeptide with a test agent, and determining the effect, if any, ofthe test agent on the expression of said fusion polypeptide from saidcoding sequence.